CN110914431A - Artificially manipulated immune cells - Google Patents

Artificially manipulated immune cells Download PDF

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CN110914431A
CN110914431A CN201880045774.2A CN201880045774A CN110914431A CN 110914431 A CN110914431 A CN 110914431A CN 201880045774 A CN201880045774 A CN 201880045774A CN 110914431 A CN110914431 A CN 110914431A
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gene
sequence
nucleic acid
immune cell
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CN110914431B (en
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金奭中
金润荣
柳浩成
郑仁英
李贞慜
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Toolgen Inc
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Toolgen Inc
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Abstract

The present invention relates to immune cell manipulation compositions for the manual manipulation of immune cells. More particularly, the present invention relates to immune cell-manipulated compositions for the manual manipulation of immune cells, comprising artificially modified immune regulatory genes and artificial receptors, as well as the production of manipulated immune cells using the compositions and uses thereof.

Description

Artificially manipulated immune cells
Technical Field
The present invention relates to the manual manipulation or modification of immunomodulatory genes. More particularly, the present invention relates to gene manipulation compositions for the manual manipulation of immunomodulatory genes and immune cells comprising the same.
Background
A cell therapeutic agent is a drug that uses living cells to induce regeneration to repair damaged or diseased cells/tissues/entities, which is a drug produced by physical, chemical, or biological manipulation (e.g., ex vivo culture, proliferation, selection, etc. of autologous cells, allogeneic cells, or xenogeneic cells).
Among them, an immunomodulatory cell therapeutic agent is a drug that achieves the purpose of disease treatment by modulating immune response in vivo using immune cells (e.g., dendritic cells, natural killer cells, T cells, etc.).
Currently, immunomodulatory cell therapeutics are being developed that mainly target cancer therapy as an indication. Unlike surgical therapies, anticancer agents and radiation therapies conventionally used for cancer treatment, immunomodulatory cell therapeutics have therapeutic mechanisms and efficacies in activating immune functions by directly administering immune cells to patients, thereby obtaining therapeutic effects; immunomodulatory cell therapeutics are expected to play an important role in emerging biology in the future.
The physical and chemical properties of antigens introduced into cells are different from each other according to the types of immunomodulatory cell therapeutic agents. When a foreign gene is introduced into immune cells in the form of a viral vector or the like, these cells will be able to have both the characteristics of a cell therapeutic agent and a gene therapeutic agent.
Administration of the immunomodulatory cell therapeutic can be carried out as follows: activating various immune cells (e.g., Peripheral Blood Mononuclear Cells (PBMCs), T cells, NK cells, etc., isolated from a patient by apheresis) by using various antibodies and cytokines, followed by ex vivo proliferation and re-injection into the patient; or the immune cells into which a gene, such as a T Cell Receptor (TCR) or a Chimeric Antigen Receptor (CAR), is introduced are re-injected into the patient.
Adoptive immunotherapy, which involves the delivery of autologous antigen-specific immune cells (e.g., T cells) generated ex vivo (ex vivo), may be a promising strategy for treating a variety of immune diseases as well as cancer.
Recently, it has been reported that immune cell therapeutic agents can be used in various ways, for example, as an autoimmune inhibitor and the like and exhibit anticancer functions. Thus, immune cell therapeutics can be used in a variety of indications by modulating the immune response. Therefore, there is a great need to develop and improve the therapeutic efficacy of the manipulated immune cells for adoptive immunotherapy.
Disclosure of Invention
Technical problem
As an exemplary embodiment, the present invention provides a composition for manipulation of immune cells for manual manipulation of immune cells.
As an exemplary embodiment, the present invention provides a manipulated immune cell comprising at least one artificially modified immunomodulatory gene and at least one artificial receptor.
As an exemplary embodiment, the present invention provides a method for producing an artificial immune cell comprising at least one artificially modified immunomodulatory gene and at least one artificial receptor.
As an exemplary embodiment, the present invention provides a method for treating an immune disease, the method comprising an artificial immune cell comprising at least one artificial modified immune regulatory gene and at least one artificial receptor as active ingredients.
Technical scheme
To address these problems, the present invention relates to compositions for manipulating immune cells. More specifically, the present invention relates to compositions for manipulating immune cells, which compositions are useful for the manual manipulation of immune cells; and manipulated immune cells comprising an artificially modified immunomodulatory gene and an artificial receptor produced using the composition, and uses thereof.
The present invention provides compositions for the manipulation of immune cells for specific purposes.
The term "composition for manipulating immune cells" refers to one or more substances selected from DNA, RNA, nucleic acids, proteins, viruses, chemical compounds, etc., which are used to manually manipulate or modify immune cells.
In certain embodiments, a composition for manipulating immune cells may comprise:
a guide nucleic acid capable of forming a complementary binding to a target sequence in the nucleic acid sequence of at least one immunomodulatory gene selected from the group consisting of: PD-1 gene, CTLA-4 gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene, PPP2r2d gene, TET2 gene, PSGL-1 gene, A20 gene and KDM6A gene; and
an artificial receptor, which is an artificially prepared receptor and is not a wild-type receptor.
The term "immunomodulatory gene" is intended to include any gene that directly anticipates or indirectly affects the development and performance of an immune function or response. In the present invention, an immunomodulatory gene includes any gene that directly anticipates or indirectly affects functional regulation of an immune cell, and also directly anticipates or indirectly affects functional regulation of a cell (e.g., phagocyte) interacting with the immune cell. Here, the immune regulatory gene can perform a function related to the formation and performance of an immune function or response in the form of the immune regulatory gene itself or a protein expressed by the immune regulatory gene.
The term "artificial receptor" refers to an artificially prepared functional entity that is not a wild-type receptor, which has a specific ability to recognize an antigen and perform a specific function.
The composition for manipulating immune cells may optionally further comprise at least one editing protein selected from the group consisting of: cas9 protein derived from Streptococcus pyogenes (Streptococcus pyogenenes), Cas9 protein derived from Campylobacter jejuni (Campylobacter jejuni), Cas9 protein derived from Streptococcus thermophilus (Streptococcus thermophilus), Cas9 protein derived from staphylococcus aureus (Streptococcus aureus), Cas9 protein derived from Neisseria meningitidis (Neisseria meningitidis), and Cpf1 protein.
The target sequence may be a contiguous 10bp-25bp nucleotide sequence located in the promoter region of the immune regulatory gene.
The target sequence may be a contiguous 10bp-25bp nucleotide sequence located in an intron region of the immunomodulatory gene.
The target sequence may be a contiguous 10bp-25bp nucleotide sequence located in an exon region of the immune regulatory gene.
The target sequence may be a contiguous 10bp-25bp nucleotide sequence located in the enhancer region of the immune modulatory gene.
The target sequence may be a contiguous 10bp-25bp nucleotide sequence located in the 3'-UTR (untranslated region) or 5' -UTR of an immunomodulatory gene.
The target sequence may be a contiguous 10bp-25bp nucleotide sequence adjacent to the 5 'end and/or 3' end of a PAM (pro-spacer sequence adjacent motif) sequence in the nucleic acid sequence of the immune modulatory gene.
Here, the PAM sequence may be at least one sequence selected from the following sequences:
5'-NGG-3' (N is A, T, C or G);
5 '-NNRYAC-3' (N is each independently A, T, C or G; R is A or G; Y is C or T);
5'-NNAGAAW-3' (N is each independently A, T, C or G; W is A or T);
5'-NNNNGATT-3' (N is each independently A, T, C or G);
5'-NNGRR (T) -3' (N is each independently A, T, C or G; R is A or G; Y is C or T); and
5'-TTN-3' (N is A, T, C or G).
In certain embodiments, the target sequence may be a sequence selected from SEQ ID NOs: 1-SEQ ID NO: 289.
The guide nucleic acid may comprise a guide domain capable of forming a complementary binding with a target sequence on an immunomodulatory gene, wherein the complementary binding may comprise 0-5 mismatches.
Here, the targeting domain may comprise a nucleotide sequence complementary to a target sequence on the immunomodulatory gene, wherein the complementary nucleotide sequence may comprise 0-5 mismatches.
The guide nucleic acid may comprise at least one domain selected from the group consisting of: a first complementary domain, a linker domain, a second complementary domain, a proximal domain, and a tail domain.
The artificial receptor may have a binding specificity for at least one antigen.
Here, the at least one antigen may be an antigen specifically expressed by cancer cells and/or viruses.
Here, the at least one antigen may be a tumor-associated antigen.
The at least one antigen may be selected from the group consisting of human chorionic gonadotropin B7H (CD276), BST, BRAP, CD44v, CD79, CD123, CD138, CD160, CD171, CD179, Carbonic Anhydrase (CAIX), CA-125, carcinoembryonic antigen (CEA), C-type lectin-like molecule (NYL-1 or CLECL), Claudin (CLD), CXORF, CAGE, CDX, CT-7, TEM-7, HOM-85, TAGE-1, ERBB, epidermal growth factor receptor (NYNYNYLB III), epidermal growth factor receptor (MAG-III), TNF-2-receptor (MAG-1-CSF-7, VEGF-2-receptor (MAG-2), VEGF-receptor (MAG-2), VEGF-2-receptor (MAG-2), VEGF-2-receptor-TNF-2-receptor (MAG-2), VEGF-2-receptor-2-TNF-2-receptor (MAG-2), VEGF-2-receptor-7, VEGF-2-receptor (MAG-2-receptor), VEGF-2-receptor-beta-receptor, VEGF-2-receptor-beta-5, VEGF-beta-receptor-5, VEGF-TNF-alpha-beta-alpha.
The artificial receptor can be a Chimeric Antigen Receptor (CAR).
The artificial receptor may be a T Cell Receptor (TCR) that is manually manipulated or modified.
The guide nucleic acid, artificial receptor, and editing protein may be in the form of nucleic acid sequences encoding each of them.
The nucleic acid sequence may be contained in a plasmid or viral vector.
Here, the viral vector may be one or more selected from the group consisting of: retrovirus, lentivirus, adenovirus, adeno-associated virus (AAV), vaccinia virus, poxvirus, or herpes simplex virus.
The artificial receptor and the editing protein may be in the form of mrnas encoding each of them.
The artificial receptor and the editing protein may be in the form of a polypeptide or a protein.
When the composition for manipulating immune cells optionally further comprises an editing protein, the composition may be in the form of a guide nucleic acid-editing protein complex.
The present invention provides manipulated immune cells for specific purposes.
"manipulated immune cells" refers to immune cells that are manipulated by man and not wild-type.
In certain embodiments, the manipulated immune cell may comprise at least one artificially engineered immune modulatory gene and/or a product expressed by said artificially engineered immune modulatory gene selected from the group consisting of: PD-1 gene, CTLA-4 gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene, PPP2r2d gene, TET2 gene, PSGL-1 gene, A20 gene and KDM6A gene; and at least one artificial receptor protein and/or a nucleic acid encoding the artificial receptor protein.
The at least one artificially engineered immunomodulatory gene may comprise an artificial modification within the nucleotide sequence of the immunomodulatory gene.
The at least one artificially engineered immunomodulatory gene may comprise a deletion and/or insertion of at least one nucleotide in a region of a 1bp-50bp nucleotide sequence within or adjacent to the 5 'end and/or 3' end of the target sequence in the immunomodulatory gene.
The at least one artificially engineered immunomodulatory gene may comprise a deletion and/or insertion of at least one nucleotide from a contiguous region of nucleotide sequences from 1bp to 50bp adjacent to the 5 'end and/or 3' end of a PAM sequence in the nucleic acid sequence of the immunomodulatory gene.
Here, the deletion of at least one nucleotide may be a continuous deletion of 1bp to 50bp, a discontinuous deletion of 1bp to 50bp, or a deletion of 1bp to 50bp in which a continuous form and a discontinuous form are mixed.
Here, the deletion of at least one nucleotide may be a continuous deletion of 2bp to 50 bp.
Here, the insertion of at least one nucleotide may be a continuous 1bp-50bp insertion, a discontinuous 1bp-50bp insertion, or a 1bp-50bp insertion in which a continuous form and a discontinuous form are mixed.
Here, the insertion of at least one nucleotide may be the insertion of a contiguous 5bp-1000bp nucleotide fragment.
Here, the insertion of at least one nucleotide may be an insertion of a partial or entire nucleotide sequence of a specific gene.
The specific gene may be an exogenous gene introduced from an external region, which is not contained in the immune cell containing the immunomodulatory gene.
The specific gene may be an endogenous gene present in the genome of an immune cell comprising the immunomodulatory gene.
Here, the deletion and insertion of at least one nucleotide may occur in the same nucleotide sequence region.
Here, the deletion and insertion of at least one nucleotide may occur in different nucleotide sequence regions.
At least one product expressed by the engineered immunomodulatory gene may be in the form of mRNA and/or protein.
The product expressed by the engineered immunomodulatory gene may have a reduced or suppressed expression level as compared to the amount of the product expressed by an immunomodulatory gene of a wild-type immune cell that has not been artificially manipulated.
Here, the wild-type immune cell that is not artificially manipulated may be an immune cell isolated from a human.
Here, the wild-type immune cell that is not manually manipulated may be an immune cell before manual manipulation.
Nucleic acids encoding the artificial receptor proteins are present in the cell, but may not be inserted into the genome of the manipulated immune cell.
Nucleic acids encoding artificial receptor proteins can be inserted into the 3'-UTR, 5' -UTR, intron, exon, promoter and/or enhancer regions of immune regulatory genes in the genome of the manipulated immune cells.
The nucleic acid encoding the artificial receptor protein may be inserted into at least one intron selected from the group consisting of introns present in the genome of the manipulated immune cell.
The nucleic acid encoding the artificial receptor protein may be inserted into at least one exon selected from exons present in the genome of the manipulated immune cell.
The nucleic acid encoding the artificial receptor protein may be inserted into at least one promoter selected from the group consisting of promoters present in the genome of the manipulated immune cell.
The nucleic acid encoding the artificial receptor protein may be inserted into at least one enhancer selected from the group consisting of enhancers present in the genome of the manipulated immune cell.
The nucleic acid encoding the artificial receptor protein may be inserted into one or more regions other than introns, exons, promoters and enhancers present in the genome of the manipulated immune cell.
The manipulated immune cell may be a manually manipulated immune cell selected from the group consisting of: dendritic cells, T cells, NK cells, NKT cells and CIK cells.
The present invention provides for a manipulated immune cell for a specific purpose, said immune cell exhibiting at least one characteristic.
In certain embodiments, the at least one feature may be one or more selected from the group consisting of:
increased production and/or secretion of cytokines;
cell proliferation, and
increased cytotoxicity.
Here, the cytokine may be one or more selected from the group consisting of IL-2, TNF α and IFN-gamma.
The explanations relating to the manipulated immune cells are as described above.
The present invention provides methods for producing manipulated immune cells for specific purposes.
In certain embodiments, a method for producing a manipulated immune cell can comprise contacting:
(a) an immune cell;
(b) an artificial receptor protein or a composition for expressing an artificial receptor protein; and
(c) a composition for gene manipulation capable of manual manipulation of at least one immunomodulatory gene selected from the group consisting of: PD-1 gene, CTLA-4 gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene, PPP2r2d, TET2 gene, PSGL-1 gene, A20 gene and KDM6A gene.
(a) The immune cell may be an immune cell isolated from a human body or an immune cell differentiated from a stem cell.
(b) Compositions for expressing an artificial receptor protein may comprise a nucleic acid sequence encoding the artificial receptor protein.
(c) Compositions for gene manipulation may comprise:
a guide nucleic acid that hybridizes with a target sequence of SEQ id no: 1-SEQ ID NO: 289 have homology or are capable of forming complementary binding therewith: PD-1 gene, CTLA-4 gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene, PPP2r2d gene, TET2 gene, PSGL-1 gene, A20 gene and KDM6A gene; and
at least one editing protein or a nucleic acid encoding the editing protein, the at least one editing protein selected from the group consisting of: a Cas9 protein derived from streptococcus pyogenes, a Cas9 protein derived from campylobacter jejuni, a Cas9 protein derived from streptococcus thermophilus, a Cas9 protein derived from staphylococcus aureus, a Cas9 protein derived from neisseria meningitidis, and a Cpf1 protein.
Here, the guide nucleic acid and the editing protein may each be in the form of a nucleic acid sequence in at least one vector, or may be in the form of a guide nucleic acid-editing protein complex in which the guide nucleic acid and the editing protein are bound.
The contacting can be performed ex vivo.
The contacting may be sequentially or simultaneously contacting (a) the immune cell with (b) a composition for expressing an artificial receptor protein and (c) a composition for gene manipulation.
The contacting may be performed by at least one method selected from the following methods: electroporation, liposomes, plasmids, viral vectors, nanoparticles, and Protein Translocation Domain (PTD) fusion protein methods.
The present invention provides methods for treating immune diseases using manipulated immune cells for specific purposes.
In certain embodiments, the method for treating an immune disease comprises administering to a subject a pharmaceutical composition comprising the manipulated immune cells as an active ingredient.
The explanations relating to the manipulated immune cells are as described above.
The pharmaceutical composition may further comprise additional components.
Here, the additional component may be an immune checkpoint inhibitor.
The immune checkpoint inhibitor may be an inhibitor of PD-1, PD-L1, LAG-3, TIM-3, CTLA-4, TIGIT, BTLA, IDO, VISTA, ICOS, KIR, CD160, CD244 or CD 39.
Here, the additional component may be an antigen binding agent, a cytokine, a secretagogue for a cytokine, or an inhibitor of a cytokine.
Here, the additional component may be a suitable vehicle for delivering the manipulated immune cells into the body.
The manipulated immune cells included in the pharmaceutical composition may be autologous cells, or allogeneic cells of the subject.
The immune disease may be an autoimmune disease.
Here, the autoimmune disease may be Graft Versus Host Disease (GVHD), systemic lupus erythematosus, celiac disease, type 1 diabetes, graves' disease, inflammatory bowel disease, psoriasis, rheumatoid arthritis, or multiple sclerosis.
The disease may be a refractory disease in which the pathogen is known but the treatment is unknown.
Here, the intractable disease may be a virus infection disease, a disease caused by prion pathogen, or cancer.
Administration of the pharmaceutical composition to a subject having an immune disorder can be performed by a method selected from injection, infusion, implantation, or transplantation.
The subjects are mammals, including humans, monkeys, mice, and rats.
Drawings
Fig. 1 to 27 illustrate examples of artificially modified or manipulated target genes.
Figure 28 is a graph showing CAR expression levels in 139CAR-T cells treated with CRISPR/Cas 9.
Fig. 29 is a graph showing T cell growth after electroporation of the CRISPR/Cas9 complex.
Fig. 30 shows DGK selective knockdown by CRISPR/Cas9 in T cells, where the graph confirms insertion deletion (%) of DGK (a) and protein expression of DGK (B).
FIG. 31 is a graph illustrating off-target sites of gRNAs for each DGK identified using digomer-Seq.
Figure 32 is a graph showing the cytotoxic effect of 139CAR-T cells, wherein AAVS1 is knocked out by CRISPR/Cas 9.
Figure 33 is a graph comparing the cytotoxic effects (a) and cytokine secretion levels (B) of 139CAR-T cells, either AAVS 1-knockout 139CAR-T cells or DGK-knockout 139CAR-T cells by CRISPR/Cas 9.
FIG. 34 is a graph comparing PDL-1 expression level (A) of U87vIII and PD-1 expression level (B) of T cells after co-culture of U87 cells or U87vIII cells with 139CAR-T cells.
Fig. 35 is a graph showing changes in calcium influx (a) and expression of pERK protein (B) in DGK-knocked 139CAR-T cells.
Figure 36 is a graph comparing the cytotoxic effects and cytokine secretion levels of 139CAR-T cells knockout AAVS1 or 139CAR-T cells knockout DGK in the presence of immunosuppressive factors, showing the presence of TGF- β (a) and PEG2 (B), respectively.
Figure 37 is a graph showing effector function of 139CAR-T cells knockout of AAVS1 or 139CAR-T cells knockout of DGK in the presence of immunosuppressive factors.
Fig. 38 is a graph showing effector function of AAVS1 knockout c259 TCR T cells or DGK knockout c259 TCR T cells in the presence of immunosuppressive factors.
Figure 39 illustrates the experimental design used to identify effector activity of DGK knockout T cells under repeated antigen exposure.
FIG. 40 illustrates cell proliferation of DGK knocked out T cells under repeated antigen exposure, where the graph compares (A) the number of viable cells and (B) the number of proliferating cells (%).
FIG. 41 illustrates Fas-mediated activity inducing cell death in DGK knocked-out T cells, where the graph shows the expression levels (%) of (A) activation-induced cell death (AICD,%) and (B) Fas.
Figure 42 is a graph showing cytokine secretion levels of 139CAR-T cells knockout AAVS1 or 139CAR-T cells knockout DGK, followed by repeated tumor vaccinations.
Fig. 43 is a graph showing (a) the amount of naive T cells and (B) the amount of effector memory T cells for either AAVS1 knockout 139CAR-T cells or DGK knockout 139CAR-T cells.
Figure 44 is a graph showing (a) expression levels of effector memory regulatory factors, (B) expression levels of type 1 cytokines, and (C) expression levels of type 2 cytokines for either AAVS1 knockout 139CAR-T cells or DGK knockout 139CAR-T cells.
Figure 45 is a graph showing expression levels of markers associated with T cell depletion in 139CAR-T cells knockout of AAVS1 or 139CAR-T cells knockout of DGK.
Figure 46 illustrates the anti-tumor effect of either AAVS1 knockout 139CAR-T cells or DGK knockout 139CAR-T cells, where the graphs show the anti-tumor effect at intravenous injection (a) and intratumoral injection (B), and the images compare the tumor size in each case (C).
FIG. 47 shows graphs comparing the maintenance (A, B) of AAVS 1139 CAR-T cells, α KO 139CAR-T cells, ζ KO 139CAR-T cells and dKO 139CAR-T cells injected in vivo and the tumor size (C) in each case, and graphs illustrating the number of tumor-infiltrating T cells (D) in each case.
FIG. 48 is a graph comparing in vivo injected IFN- γ, TNF α positive (%) (A), Ki-67 positive (%) (B), and T-beta positive (%) (C) of AAVS 1139 CAR-T cells, α KO 139CAR-T cells, ζ KO 139CAR-T cells, and dKO 139CAR-T cells.
Detailed Description
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
One aspect disclosed herein relates to a guide nucleic acid.
The term "guide nucleic acid" refers to a nucleotide sequence that can recognize a target nucleic acid, target gene, or target chromosome and interact with an editing protein. The guide nucleic acid can be complementarily bound to a portion of the nucleotide sequence in the target nucleic acid, target gene, or target chromosome. And, a part of the nucleotide sequence in the guide nucleic acid may interact with a part of the amino acids in the editing protein and form a guide nucleic acid-editing protein complex.
The guide nucleic acid can serve to induce the guide nucleic acid-editing protein complex to localize to a target nucleic acid, target gene, or target region of a target chromosome.
The guide nucleic acid may be present in the form of DNA, RNA or a DNA/RNA mixture and has a sequence of 5 to 150 nucleic acids.
The guide nucleic acid may be a contiguous nucleic acid sequence.
For example, the one contiguous nucleic acid sequence can be (N) m, wherein N is A, T, C or G, or A, U, C or G; m is an integer of 1 to 150.
The guide nucleic acid may be two or more contiguous nucleic acid sequences.
For example, the two or more contiguous nucleic acid sequences may be (N) m and (N) o, wherein N represents A, T, C or G, or represents A, U, C or G; m and o are integers of 1 to 150, and may be the same as or different from each other.
The guide nucleic acid comprises one or more domains.
The domain may be a functional domain such as, but not limited to, a leader domain, a first complementary domain, a linker domain, a second complementary domain, a proximal (proximal) domain, or a tail domain.
Here, one guide nucleic acid may have more than two functional domains. Furthermore, more than two functional domains may be different from each other. Alternatively, two or more functional domains contained in the guide nucleic acid may be identical to each other. For example, a guide nucleic acid can have more than two proximal domains, and in another example, a guide nucleic acid can have more than two tail domains. However, the functional domains contained in the guide nucleic acid being two identical domains does not mean that the two functional domains have the same sequence; although the sequences of the domains are different, they are considered to be identical as long as they perform the same function.
Details regarding the functional domains are detailed below.
i) Leader Domain
The term "guide domain" is a domain having a complementary guide sequence capable of forming a complementary binding with a partial sequence in a target gene or nucleic acid, and functions to specifically interact with the target gene or nucleic acid. For example, the guide domain can be used to induce a guide nucleic acid-editing protein complex to a location having a specific nucleotide sequence of a target gene or nucleic acid.
The leader domain may be a nucleotide sequence of 10bp to 35 bp.
In one example, the guide domain can be a nucleotide sequence of 10bp-35bp, 15bp-35bp, 20bp-35bp, 25bp-35bp, or 30bp-35 bp.
In another example, the guide domain can be a nucleotide sequence of 10bp-15bp, 15bp-20bp, 20bp-25bp, 25bp-30bp, or 30bp-35 bp.
The leader domain may comprise a leader sequence.
The term "guide sequence" is a nucleotide sequence that is complementary to a partial sequence in one strand of a duplex of a target gene or nucleic acid, wherein the guide sequence can be a nucleotide sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more complementarity or complete complementarity.
The guide sequence may be a nucleotide sequence of 10bp to 25 bp.
In one example, the leader sequence may be a nucleotide sequence of 10bp to 25bp, 15bp to 25bp, or 20bp to 25 bp.
In another example, the leader sequence may be a nucleotide sequence of 10bp to 15bp, 15bp to 20bp, or 20bp to 25 bp.
In addition, the guide domain may have an additional nucleotide sequence.
The additional nucleotide sequence may be a sequence that promotes or inhibits the function of the leader domain.
The additional nucleotide sequence may be a sequence that promotes or inhibits the function of the leader sequence.
The additional nucleotide sequence may be a nucleotide sequence of 1bp to 10 bp.
In one example, the additional nucleotide sequence can be a nucleotide sequence of 2bp-10bp, 4bp-10bp, 6bp-10bp, or 8bp-10 bp.
In another example, the additional nucleotide sequence may be a nucleotide sequence of 1bp-3bp, 3bp-6bp, or 7bp-10 bp.
In embodiments, the additional nucleotide sequence may be a 1bp, 2bp, 3bp, 4bp, 5bp, 6bp, 7bp, 8bp, 9bp, or 10bp nucleotide sequence.
For example, the additional nucleotide sequence may be a1 base nucleotide sequence G (guanine) or a2 base nucleotide sequence GG.
The additional nucleotide sequence may be located 5' to the leader sequence.
The additional nucleotide sequence may be located 3' to the leader sequence.
ii) a first complementary domain
The term "first complementary domain" is a domain comprising a nucleotide sequence complementary to a second complementary domain, explained below, which has sufficient complementarity to form a double strand with the second complementary domain. For example, the first complementing domain may be a nucleotide sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more complementarity or complete complementarity to the second complementing domain.
The first complementary domain can form a double strand with the second complementary domain by complementary binding. The duplex can be used to interact with a portion of the amino acids in the editing protein, thereby forming a guide nucleic acid-editing protein complex.
The first complementary domain can be a sequence of 5-35 nucleotides.
In one example, the first complementary domain can be a sequence of 5-35 nucleotides, 10-35 nucleotides, 15-35 nucleotides, 20-35 nucleotides, 25-35 nucleotides, or 30-35 nucleotides.
In another example, the first complementary domain can be a sequence of 1-5 nucleotides, 5-10 nucleotides, 10-15 nucleotides, 15-20 nucleotides, 20-25 nucleotides, 25-30 nucleotides, or 30-35 nucleotides.
iii) linker Domain
The term "linker domain" is a nucleic acid sequence that links two or more domains (two or more identical or different domains). The linker domain may be linked to the two or more domains by covalent or non-covalent bonds, or the two or more domains may be linked by covalent or non-covalent bonds.
The linker domain may be a sequence of 1-30 nucleotides.
In one example, the linker domain can be a sequence of 1-5 nucleotides, 5-10 nucleotides, 10-15 nucleotides, 15-20 nucleotides, 20-25 nucleotides, or 25-30 nucleotides.
In another example, the linker domain can be a sequence of 1-30 nucleotides, 5-30 nucleotides, 10-30 nucleotides, 15-30 nucleotides, 20-30 nucleotides, or 25-30 nucleotides.
iv) a second complementary domain
The term "second complementary domain" is a domain comprising a nucleotide sequence, said domain comprising a nucleic acid sequence complementary to said first complementary domain as described above, with sufficient complementarity to form a double strand with the first complementary domain. For example, the second complementary domain can be a nucleotide sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more complementarity or complete complementarity to the first complementary domain.
The second complementary domain can form a double strand with the first complementary domain by complementary binding. The double strand formed can be used to interact with a portion of the amino acids in the editing protein, thereby forming a guide nucleic acid-editing protein complex.
The second complementary domain can have a nucleotide sequence that is complementary to the first complementary domain and a nucleotide sequence that is not complementary to the first complementary domain (e.g., a nucleotide sequence that does not form a double strand with the first complementary domain) and can have a longer nucleotide sequence than the first complementary domain.
The second complementary domain can have a sequence of 5-35 nucleotides.
In an example, the second complementary domain can be a sequence of 1-35 nucleotides, 5-35 nucleotides, 10-35 nucleotides, 15-35 nucleotides, 20-35 nucleotides, 25-35 nucleotides, or 30-35 nucleotides.
In another example, the second complementary domain can be a sequence of 1-5 nucleotides, 5-10 nucleotides, 10-15 nucleotides, 15-20 nucleotides, 20-25 nucleotides, 25-30 nucleotides, or 30-35 nucleotides.
v) a proximal domain
The term "proximal domain" is a nucleotide sequence that is located proximal to the second complementary domain.
The proximal domain may have a complementary nucleotide sequence, and may form a double strand based on the complementary nucleotide sequence.
The proximal domain may be a sequence of 1-20 nucleotides.
In one example, the proximal domain can be a sequence of 1-20 nucleotides, 5-20 nucleotides, 10-20 nucleotides, or 15-20 nucleotides.
In another example, the proximal domain can be a sequence of 1-20 bases, 5-20 bases, 10-20 bases, or 15-20 bases. The proximal domain may be a sequence of 1-5 nucleotides, 5-10 nucleotides, 10-15 nucleotides, or 15-20 nucleotides.
vi) the Tail Domain
The term "tail domain" is a nucleotide sequence located at one or more of the two ends of the guide nucleic acid.
The tail domain may have a complementary nucleotide sequence and may form a double strand based on the complementary nucleotide sequence.
The tail domain may be a sequence of 1-50 nucleotides.
In examples, the tail domain may be a sequence of 5-50 nucleotides, 10-50 nucleotides, 15-50 nucleotides, 20-50 nucleotides, 25-50 nucleotides, 30-50 nucleotides, 35-50 nucleotides, 40-50 nucleotides, or 45-50 nucleotides.
In another example, the tail domain can be a sequence of 1-5 nucleotides, 5-10 nucleotides, 10-15 nucleotides, 15-20 nucleotides, 20-25 nucleotides, 25-30 nucleotides, 30-35 nucleotides, 35-40 nucleotides, 40-45 nucleotides, or 45-50 nucleotides.
Meanwhile, part or all of the nucleic acid sequences contained in the domains (i.e., the guide domain, the first complementary domain, the linker domain, the second complementary domain, the proximal domain, and the tail domain) may optionally or additionally contain chemical modifications.
The chemical modification may be, but is not limited to, methylation, acetylation, phosphorylation, phosphorothioate linkages, Locked Nucleic Acids (LNA), 2 '-O-methyl 3' phosphorothioate (MS), or 2 '-O-methyl 3' thiopace (msp).
The guide nucleic acid comprises one or more domains.
The guide nucleic acid may comprise a guide domain.
The guide nucleic acid may comprise a first complementary domain.
The guide nucleic acid may comprise a linker domain.
The guide nucleic acid may comprise a second complementary domain.
The guide nucleic acid may comprise a proximal domain.
The guide nucleic acid may comprise a tail domain.
Here, more than 1, 2, 3, 4, 5, 6 domains may be present.
The guide nucleic acid may comprise more than 1, 2, 3, 4, 5, 6 guide domains.
The guide nucleic acid may comprise more than 1, 2, 3, 4, 5, 6 first complementary domains.
The guide nucleic acid may comprise more than 1, 2, 3, 4, 5, 6 linker domains.
The guide nucleic acid may comprise more than 1, 2, 3, 4, 5, 6 second complementary domains.
The guide nucleic acid may comprise more than 1, 2, 3, 4, 5, 6 proximal domains.
The guide nucleic acid may comprise more than 1, 2, 3, 4, 5, 6 tail domains.
Here, in the guide nucleic acid, one type of domain may be repeated.
The guide nucleic acid may comprise several domains with or without repeats.
The guide nucleic acid may comprise domains of the same type. Here, the same type of domain may have the same nucleic acid sequence or different nucleic acid sequences.
The guide nucleic acid may comprise two types of domains. Here, the two different types of domains may have different nucleic acid sequences or the same nucleic acid sequence.
The guide nucleic acid may comprise three types of domains. Here, the three different types of domains may have different nucleic acid sequences or the same nucleic acid sequence.
The guide nucleic acid may comprise four types of domains. Here, the four different types of domains may have different nucleic acid sequences or the same nucleic acid sequence.
The guide nucleic acid may comprise five types of domains. Here, the five different types of domains may have different nucleic acid sequences or the same nucleic acid sequence.
The guide nucleic acid may comprise six types of domains. Here, the six different types of domains may have different nucleic acid sequences or the same nucleic acid sequence.
For example, the guide nucleic acid can be comprised of [ guide domain ] - [ first complementary domain ] - [ linker domain ] - [ second complementary domain ] - [ linker domain ] - [ guide domain ] - [ first complementary domain ] - [ linker domain ] - [ second complementary domain ]. Here, the two targeting domains may comprise targeting sequences for different or the same targets; the two first and second complementary domains may have the same or different nucleic acid sequences. When the guide domain comprises guide sequences for different targets, the guide nucleic acid can specifically bind to two different targets; here, the specific binding may be performed simultaneously or sequentially. In addition, the linker domain may be cleaved by a particular enzyme, and the guide nucleic acid may be divided into two or three portions in the presence of the particular enzyme.
As an embodiment of the disclosure herein, the guide nucleic acid may be a gRNA.
gRNA
The term "gRNA" refers to a nucleic acid capable of specifically directing a gRNA-CRISPR enzyme complex (i.e., CRISPR complex) to a target gene or nucleic acid. Further, a gRNA is a nucleic acid-specific RNA that can bind to and direct a CRISPR enzyme to a target gene or nucleic acid.
grnas may comprise multiple domains. Based on the individual domains, interactions can occur in or between the chains of the three-dimensional structure or active form of the gRNA.
A gRNA may refer to a single-stranded gRNA (a single RNA molecule) or a double-stranded gRNA (comprising more than one RNA molecule, typically two separate RNA molecules).
In one exemplary embodiment, a single-stranded gRNA can comprise, in a5 'to 3' direction, a guide domain (i.e., a domain comprising a guide sequence capable of forming a complementary binding with a target gene or nucleic acid), a first complementary domain, a linker domain, a second complementary domain (having a sequence complementary to the sequence of the first complementary domain, thereby forming a double-stranded nucleic acid with the first complementary domain), a proximal domain, and optionally a tail domain.
In another embodiment, a double-stranded gRNA can comprise a first strand comprising a guide domain (i.e., a domain comprising a guide sequence capable of forming a complementary binding with a target gene or nucleic acid) and a first complementary domain; the second strand comprises, in the 5 'to 3' direction, a second complementary domain (which has a sequence complementary to the sequence of the first complementary domain, thus forming a double-stranded nucleic acid with the first complementary domain), a proximal domain, and optionally a tail domain.
Here, the first strand may be referred to as crRNA and the second strand may be referred to as tracrRNA. The crRNA may comprise a guide domain and a first complementary domain; the tracrRNA may comprise a second complementary domain, a proximal domain, and optionally a tail domain.
In yet another embodiment, the single-stranded gRNA may comprise, in a3 'to 5' direction, a guide domain (i.e., a domain comprising a guide sequence capable of forming a complementary binding with a target gene or nucleic acid), a first complementary domain, and a second complementary domain (the domains having a sequence complementary to the sequence of the first complementary domain, thereby forming a double-stranded nucleic acid with the first complementary domain).
The first complementary domain may have homology to the native first complementary domain or may be derived from the native first complementary domain. In addition, the first complementary domain may differ in base sequence of the first complementary domain depending on the naturally-occurring species, may be derived from the first complementary domain contained in the naturally-occurring species, or may have partial or complete homology with the first complementary domain contained in the naturally-occurring species.
In an exemplary embodiment, the first complementary domain may have partial (i.e., at least 50% or more) or complete homology to the first complementary domain of, or derived from, streptococcus pyogenes, campylobacter jejuni, streptococcus thermophilus, staphylococcus aureus, or neisseria meningitidis.
For example, when the first complementary domain is that of streptococcus pyogenes or a first complementary domain derived therefrom, the first complementary domain can be 5'-GUUUUAGAGCUA-3' or a base sequence having partial (i.e., at least 50% or greater) or complete homology to 5'-GUUUUAGAGCUA-3'. Here, the first complementary structureThe domain may further comprise (X)nSo that it is 5' -GUUUUAGAGCUA(X)n-3'. X may be selected from the group consisting of bases A, T, U and G; n may represent the number of bases, which is an integer of 5 to 15. Here, (X)nCan be n repeats of the same base, or a mixture of n bases A, T, U and G.
In another embodiment, when the first complementary domain is a first complementary domain of campylobacter jejuni or a first complementary domain derived therefrom, the first complementary domain may be 5'-GUUUUAGUCCCUUUUUAAAUUUCUU-3' or 5'-GUUUUAGUCCCUU-3', or a base sequence having partial (i.e., at least 50% or more) or complete homology to 5'-GUUUUAGUCCCUUUUUAAAUUUCUU-3' or 5'-GUUUUAGUCCCUU-3'. Here, the first complementary domain may further comprise (X)nSo that it is 5' -GUUUUAGUCCCUUUUUAAAUUUCUU(X)n-3 'or 5' -GUUUUAGUCCCUU(X)n-3'. X may be selected from the group consisting of bases A, T, U and G; n may represent the number of bases, which is an integer of 5 to 15. Here, (X)nMay represent n repeats of the same base or a mixture of n bases A, T, U and G.
In another embodiment, the first complementing domain may have partial (i.e. at least 50% or more) or complete homology to the first complementing domain of or derived from: thrifty bacterium (Parcubibacterium) (GWC2011_ GWC2_44_17), Muospira hirsuta (Lachnospiraceae bacterium) (MC2017), Butyrivibrio proteoclasius, Peregrinibacteria (GW2011_ GWA _33_10), Aminococcus (Acidococcus sp.) (BV3L6), Porphyromonas (Porphyromonas macrocacea), Muospirillum (ND2006), Porphyromonas crevicans, Peptospira saccharolytica (Prevotella discoides), Moraxella borvaculi (237), Smiihella sp. (SC _ KO8D17), Gluconobacter canicola (Leptospira inadada), Mucospora hirsuta (Francisella), Metarhizia novaenopsis (M), and Mycobacterium tuberculosis (Metallus 112).
For example, when the first complementing domain is a first complementing domain of thrifton bacterium or a first complementing domain derived therefrom, the first complementing domainThe complementary domain can be 5 '-uuuguagauu-3' or a base sequence having partial (i.e., at least 50% or more) homology to 5 '-uuuguagauu-3'. Here, the first complementary domain may further comprise (X)nSo that it is 5' - (X)nUUGUAGAU-3'. X may be selected from the group consisting of bases A, T, U and G; n may represent the number of bases, which is an integer of 1 to 5. Here, (X)nMay represent n repeats of the same base or a mixture of n bases A, T, U and G.
Here, the linker domain may be a nucleotide sequence that links the first complementary domain with the second complementary domain.
The linker domain may be linked to the first and second complementary domains, respectively, by covalent or non-covalent bonds.
The linker domain may join the first complementary domain and the second complementary domain by covalent or non-covalent bonds.
The linker domain is suitable for use in single-stranded gRNA molecules, and can be used to join or link first and second strands of a double-stranded gRNA via covalent or non-covalent bonds to produce a single-stranded gRNA.
The linker domain can be used to link or join crRNA and tracrRNA of a double-stranded gRNA via covalent or non-covalent bonds to produce a single-stranded gRNA.
Here, the second complementary domain may have homology with the native second complementary domain, or may be derived from the native second complementary domain. In addition, the second complementary domain may differ in base sequence of the second complementary domain depending on the naturally-occurring species, may be derived from the second complementary domain contained in the naturally-occurring species, or may have partial or complete homology with the second complementary domain contained in the naturally-occurring species.
In exemplary embodiments, the second complementary domain may have partial (i.e., at least 50% or more) or complete homology to the second complementary domain of, or derived from, streptococcus pyogenes, campylobacter jejuni, streptococcus thermophilus, staphylococcus aureus, or neisseria meningitidis.
For example, when the second complementary domain is the second complementary domain of Streptococcus pyogenes or a second complementary domain derived therefrom, the second complementary domain can be 5-UAGCAAGUUAAAAU-3' or 5-UAGCAAGUUAAAAU-3' has a partial (i.e., at least 50% or more) homology (base sequence forming a double strand with the first complementary domain is underlined). Here, the second complementary domain may further comprise (X)nAnd/or (X)mSo that it is 5' - (X)n UAGCAAGUUAAAAU(X)m-3'. X may be selected from the group consisting of bases A, T, U and G; n and m may each represent the number of bases, wherein n may be an integer of 1 to 15, and m may be an integer of 1 to 6. Here, (X)nMay represent n repeats of the same base or a mixture of n bases A, T, U and G. Furthermore, (X)mCan represent m repeats of the same base, or a mixture of m bases A, T, U and G.
In another example, when the second complementary domain is a second complementary domain of Campylobacter jejuni or a second complementary domain derived therefrom, the second complementary domain can be 5-AAGAAAUUUAAAAAGGGACUAAAAU-3' or 5-AAGGGACUAAAAU-3, or with 5-AAGAAAUUUAAAAAGGGACUAAAAU-3' or 5-AAGGGACUAAAAU-3' has a partial (i.e., at least 50% or more) homology (base sequence forming a double strand with the first complementary domain is underlined). Here, the second complementary domain may further comprise (X)nAnd/or (X)mSo that it is 5' - (X)n AAGAAAUUUAAAAAGGGACUAAAAU(X)m-3 'or 5' - (X) nAAGAAAUUUAAAAAU (X) m-3'. X may be selected from the group consisting of bases A, T, U and G; n and m may each represent the number of bases, wherein n may be an integer of 1 to 15, and m may be an integer of 1 to 6. Here, (X)nMay represent n repeats of the same base or a mixture of n bases A, T, U and G. Furthermore, (X)mCan represent m repeats of the same base, or a mixture of m bases A, T, U and G.
In another embodiment, the second complementing domain may have partial (i.e. at least 50% or more) or complete homology to the first complementing domain of or a second complementing domain derived from: thrifty bacterium (Parcubibacterium) (GWC2011_ GWC2_44_17), Muospira hirsuta (Lachnospiraceae bacterium) (MC2017), Butyrivibrio proteoclasius, Peregrinibacteria (GW2011_ GWA _33_10), Aminococcus (Acidococcus sp.) (BV3L6), Porphyromonas (Porphyromonas macrocacea), Muospirillum (ND2006), Porphyromonas crevicans, Peptospira saccharolytica (Prevotella discoides), Moraxella borvaculi (237), Smiihella sp. (SC _ KO8D17), Gluconobacter canicola (Leptospira inadada), Mucospora hirsuta (Francisella), Metarhizia novaenopsis (M), and Mycobacterium tuberculosis (Metallus 112).
For example, when the second complementing domain is a second complementing domain of thrifton (R) or a second complementing domain derived therefrom, the second complementing domain may be 5' -AAAUUUCUACU-3 'or with 5' -AAAUUUCUACU-3' has a partial (i.e., at least 50% or more) homology (base sequence forming a double strand with the first complementary domain is underlined). Here, the second complementary domain may further comprise (X)nAnd/or (X)mSo that it is 5' - (X) nAAUUUCUACU (X) m-3'. X may be selected from the group consisting of bases A, T, U and G, and n and m may each represent the number of bases, where n may be an integer from 1 to 10 and m may be an integer from 1 to 6. Here, (X)nMay represent n repeats of the same base or a mixture of n bases A, T, U and G. Furthermore, (X)mCan represent m repeats of the same base, or a mixture of m bases A, T, U and G.
Here, the first complementary domain and the second complementary domain may form a complementary binding.
The first complementary domain and the second complementary domain can form a double strand by complementary binding.
The double strand formed can interact with the CRISPR enzyme.
Optionally, the first complementary domain may comprise an additional nucleotide sequence that does not form a complementary binding with the second complementary domain of the second strand.
Here, the additional nucleotide sequence may be a nucleotide sequence of 1bp to 15 bp. For example, the additional nucleotide sequence may be a nucleotide sequence of 1bp to 5bp, 5bp to 10bp, or 10bp to 15 bp.
Here, the proximal domain may be a domain of the second complementary domain in the 5 'to 3' direction.
The proximal domain may have homology to the native proximal domain or may be derived from the native proximal domain. In addition, the proximal domain may differ in base sequence depending on the naturally occurring species, may be derived from a proximal domain contained in the naturally occurring species, or may have partial or complete homology with a proximal domain contained in the naturally occurring species.
In exemplary embodiments, the proximal domain may have partial (i.e., at least 50% or more) or complete homology to the proximal domain of, or derived from, streptococcus pyogenes, campylobacter jejuni, streptococcus thermophilus, staphylococcus aureus, or neisseria meningitidis.
For example, when the proximal domain is that of streptococcus pyogenes or a proximal domain derived therefrom, the proximal domain can be 5'-AAGGCUAGUCCG-3' or a base sequence having partial (i.e., at least 50% or more) homology to 5'-AAGGCUAGUCCG-3'. Here, the proximal domain may further comprise (X)nTo make it 5' -AAGGCUAGUCCG(X)n-3'. X may be selected from the group consisting of bases A, T, U and G; n may represent the number of bases, which may be an integer of 1 to 15. Here, (X)nMay represent n repeats of the same base or a mixture of n bases A, T, U and G.
In yet another embodiment, when the proximal domain is the proximal domain of campylobacter jejuni or a proximal domain derived therefrom, the proximal domain may be 5'-AAAGAGUUUGC-3' or a base sequence having at least 50% or more homology to 5'-AAAGAGUUUGC-3'. Here, the proximal domain may further comprise (X)nTo make it 5' -AAAGAGUUUGC(X)n-3'. X may be selected from the group consisting of bases A, T, U and G; n may represent the number of bases, which mayIs an integer of 1 to 40. Here, (X)nMay represent n repeats of the same base or a mixture of n bases A, T, U and G.
Here, a tail domain may be optionally added to the 3 'end of the first strand or the 3' end of the second strand of a single-stranded gRNA or a double-stranded gRNA.
Furthermore, the tail domain may have homology to the native tail domain or may be derived from the native tail domain. In addition, the tail domain may differ in base sequence depending on the naturally occurring species, may be derived from a tail domain contained in the naturally occurring species, or may have partial or complete homology with a tail domain contained in the naturally occurring species.
In an exemplary embodiment, the tail domain may have partial (i.e., at least 50% or more) or complete homology to the tail domain of, or derived from, streptococcus pyogenes, campylobacter jejuni, streptococcus thermophilus, staphylococcus aureus, or neisseria meningitidis.
For example, when the tail domain is that of streptococcus pyogenes or derived therefrom, the tail domain may be 5'-UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3' or a base sequence having partial (i.e., at least 50% or more) homology to 5'-UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3'. Here, the tail domain may further comprise (X)nTo make it 5' -UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC(X)n-3'. X may be selected from the group consisting of bases A, T, U and G; n may represent the number of bases, which may be an integer of 1 to 15. Here, (X)nMay represent n repeats of the same base or a mixture of n bases (e.g., A, T, U and G).
In another example, when the tail domain is that of campylobacter jejuni or a tail domain derived therefrom, the tail domain can be 5'-GGGACUCUGCGGGGUUACAAUCCCCUAAAACCGCUUUU-3' or a base sequence having partial (i.e., at least 50% or more) homology to 5'-GGGACUCUGCGGGGUUACAAUCCCCUAAAACCGCUUUU-3'. Here, the tail domain may further comprise (X)nRendering it 5' -GGGACUCUGCGGGGUUACAAUCCCCUAAAACCGCUUUU(X)n-3'. X may be selected from the group consisting of bases A, T, U and G; n may represent the number of bases, which may be an integer of 1 to 15. Here, (X)nMay represent n repeats of the same base or a mixture of n bases A, T, U and G.
In another embodiment, the tail domain may comprise a sequence of 1-10 bases at the 3' end that is involved in an in vitro or in vivo transcription process.
For example, when the T7 promoter is used for in vitro transcription of grnas, the tail domain can be any base sequence present at the 3' end of the DNA template. Furthermore, when the U6 promoter is used for in vivo transcription, the tail domain may be UUUUUU; when the H1 promoter is used for transcription, the tail domain may be a uuuuuu; and when a pol-III promoter is used, the tail domain may contain several uracil bases or alternative bases.
The gRNA may comprise multiple domains as described above, and thus the length of the nucleic acid sequence may be adjusted according to the domains contained in the gRNA; based on the individual domains, interactions can occur in or between the chains of the three-dimensional structure or active form of the gRNA.
A gRNA may refer to a single-stranded gRNA (a single RNA molecule) or a double-stranded gRNA (comprising more than one RNA molecule, typically two separate RNA molecules).
Double-stranded gRNA
A double-stranded gRNA consists of a first strand and a second strand.
Here, the first chain may be composed of
5'- [ guide domain ] - [ first complementary domain ] -3'; and
the second chain can be composed of
5'- [ second complementary Domain ] - [ proximal Domain ] -3' or
5'- [ second complementary domain ] - [ proximal domain ] - [ tail domain ] -3'.
Here, the first strand may refer to crRNA, and the second strand may refer to tracrRNA.
Here, the first strand and the second strand may optionally comprise additional nucleotide sequences.
In one example, the first chain may be
5'-(NTarget)-(Q)m-3'; or
5'-(X)a-(NTarget)-(X)b-(Q)m-(X)c-3'。
Here, NTargetIs a nucleotide sequence complementary to a partial sequence in one strand of the double strand of the target gene or nucleic acid, and is a nucleotide sequence region that can be changed depending on the target sequence on the target gene or nucleic acid.
Here, (Q)mIs a base sequence comprising a first complementary domain capable of forming a complementary binding with a second complementary domain of a second strand. (Q)mMay be a sequence having partial or complete homology to a first complementary domain of a naturally occurring species; the base sequence of the first complementary domain may be altered depending on the species of origin. Q may each be independently selected from the group consisting of A, U, C and G; m may be the number of bases, which is an integer of 5 to 35.
For example, when the first complementary domain has partial or complete homology with the first complementary domain of Streptococcus pyogenes or the first complementary domain derived from Streptococcus pyogenes, (Q)mMay be 5'-GUUUUAGAGCUA-3' or a base sequence having at least 50% or more homology to 5'-GUUUUAGAGCUA-3'.
In another example, when the first complementary domain has partial or complete homology with the first complementary domain of Campylobacter jejuni or the first complementary domain derived from Campylobacter jejuni, (Q)mMay be 5'-GUUUUAGUCCCUUUUUAAAUUUCUU-3' or 5'-GUUUUAGUCCCUU-3', or a base sequence having at least 50% or more homology to 5'-GUUUUAGUCCCUUUUUAAAUUUCUU-3' or 5'-GUUUUAGUCCCUU-3'.
In yet another example, (Q) when the first complementing domain has partial or complete homology with the first complementing domain of S.thermophilus or the first complementing domain derived from S.thermophilusmMay be 5'-GUUUUAGAGCUGUGUUGUUUCG-3' or a base sequence having at least 50% or more homology to 5'-GUUUUAGAGCUGUGUUGUUUCG-3'.
Furthermore, (X)a、(X)b、(X)cEach is an optional additional base sequence, wherein each X may be independently selected from the group consisting of A, U, C and G; a. b and c may each be a number of bases which is 0 or an integer of 1 to 20.
In one exemplary embodiment, the second chain can be 5' - (Z)h-(P)k-3'; or 5' - (X)d-(Z)h-(X)e-(P)k-(X)f-3'。
In another embodiment, the second chain can be 5' - (Z)h-(P)k-(F)i-3'; or 5' - (X)d-(Z)h-(X)e-(P)k-(X)f-(F)i-3'。
Here, (Z)hIs a base sequence comprising a second complementary domain capable of forming a complementary binding with the first complementary domain of the first strand. (Z)hMay be a sequence having partial or complete homology to a second complementary domain of a naturally occurring species; the base sequence of the second complementary domain may be modified depending on the species of origin. Each Z may be independently selected from the group consisting of A, U, C and G; h may be the number of bases, which may be an integer of 5 to 50.
For example, when the second complementary domain has partial or complete homology with a second complementary domain of Streptococcus pyogenes or a second complementary domain derived from Streptococcus pyogenes, (Z)hMay be 5'-UAGCAAGUUAAAAU-3' or a base sequence having at least 50% or more homology to 5'-UAGCAAGUUAAAAU-3'.
In another example, when the second complementary domain has partial or complete homology with the second complementary domain of Campylobacter jejuni or the second complementary domain derived from Campylobacter jejuni, (Z)hMay be 5'-AAGAAAUUUAAAAAGGGACUAAAAU-3' or 5'-AAGGGACUAAAAU-3', or a base sequence having at least 50% or more homology to 5'-AAGAAAUUUAAAAAGGGACUAAAAU-3' or 5'-AAGGGACUAAAAU-3'.
In yet another example, when the second complementing domain is identical to the second complementing domain of S.thermophilusOr a second complementary domain derived from S.thermophilus, having partial or complete homology (Z)hMay be 5'-CGAAACAACACAGCGAGUUAAAAU-3' or a base sequence having at least 50% or more homology to 5'-CGAAACAACACAGCGAGUUAAAAU-3'.
(P)kIs a base sequence comprising a proximal domain, which may have partial or complete homology with the proximal domain of a naturally occurring species; the base sequence of the proximal domain may be modified depending on the species of origin. Each P may be independently selected from the group consisting of A, U, C and G; k may be the number of bases, which is an integer of 1 to 20.
For example, when the proximal domain has partial or complete homology with the proximal domain of Streptococcus pyogenes or a proximal domain derived from Streptococcus pyogenes, (P)kMay be 5'-AAGGCUAGUCCG-3' or a base sequence having at least 50% or more homology to 5'-AAGGCUAGUCCG-3'.
In another example, when the proximal domain has partial or complete homology with the proximal domain of Campylobacter jejuni or the proximal domain derived from Campylobacter jejuni, (P)kMay be 5'-AAAGAGUUUGC-3' or a base sequence having at least 50% or more homology to 5'-AAAGAGUUUGC-3'.
In yet another example, when the proximal domain has partial or complete homology to the proximal domain of S.thermophilus or to a proximal domain derived from S.thermophilus, (P)kMay be 5'-AAGGCUUAGUCCG-3' or a base sequence having at least 50% or more homology to 5'-AAGGCUUAGUCCG-3'.
(F)iMay be a base sequence comprising a tail domain, which may have partial or complete homology with the tail domain of a naturally occurring species; the base sequence of the tail domain may be modified depending on the species of origin. Each F may be independently selected from the group consisting of A, U, C and G; i may be the number of bases, which is an integer of 1 to 50.
For example, when the tail domain has partial or complete identity to the tail domain of Streptococcus pyogenes or a tail domain derived from Streptococcus pyogenesWhen source (F)iMay be 5'-UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3' or a base sequence having at least 50% or more homology to 5'-UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3'.
In another example, when the tail domain has partial or complete homology with the tail domain of Campylobacter jejuni or the tail domain derived from Campylobacter coli, (F)iMay be 5'-GGGACUCUGCGGGGUUACAAUCCCCUAAAACCGCUUUU-3' or a base sequence having at least 50% or more homology to 5'-GGGACUCUGCGGGGUUACAAUCCCCUAAAACCGCUUUU-3'.
In yet another example, (F) when the tail domain has partial or complete homology to the tail domain of S.thermophilus or a tail domain derived from S.thermophilusiMay be 5'-UACUCAACUUGAAAAGGUGGCACCGAUUCGGUGUUUUU-3' or a base sequence having at least 50% or more homology to 5'-UACUCAACUUGAAAAGGUGGCACCGAUUCGGUGUUUUU-3'.
Furthermore, (F)iThe 3' end may comprise a sequence of 1-10 bases involved in vitro or in vivo transcription methods.
For example, when the T7 promoter is used for in vitro transcription of grnas, the tail domain can be any base sequence present at the 3' end of the DNA template. Furthermore, when the U6 promoter is used for in vivo transcription, the tail domain may be UUUUUU; when the H1 promoter is used for in vivo transcription, the tail domain may be a uuuuu; and when a pol-III promoter is used, the tail domain may contain several uracil bases or alternative bases.
Furthermore, (X)d、(X)eAnd (X)fOptionally added base sequence, wherein each X can be independently selected from the group consisting of A, U, C and G; d. e, f each may be a number of bases which is 0 or an integer of 1 to 20.
Single-stranded gRNA
The single-stranded gRNA may be divided into a first single-stranded gRNA and a second single-stranded gRNA.
First single-stranded gRNA
The first single-stranded gRNA is a single-stranded gRNA in which a first strand and a second strand of a double-stranded gRNA are joined by a linker domain.
Specifically, the single-stranded gRNA may be composed of
5'- [ directing domain ] - [ first complementary domain ] - [ linker domain ] - [ second complementary domain ] -3',
5'- [ directing domain ] - [ first complementing domain ] - [ linker domain ] - [ second complementing domain ] - [ proximal domain ] -3'; or
5'- [ directing domain ] - [ first complementary domain ] - [ linker domain ] - [ second complementary domain ] - [ proximal domain ] - [ tail domain ] -3'.
The first single-stranded gRNA can optionally comprise additional nucleotide sequences.
In one exemplary embodiment, the first single-stranded gRNA may be a gRNA
5'-(NTarget)-(Q)m-(L)j-(Z)h-3'；
5'-(NTarget)-(Q)m-(L)j-(Z)h-(P)k-3'; or
5'-(NTarget)-(Q)m-(L)j-(Z)h-(P)k-(F)i-3'。
In another exemplary embodiment, the single-stranded gRNA can be a single-stranded gRNA
5'-(X)a-(NTarget)-(X)b-(Q)m-(X)c-(L)j-(X)d-(Z)h-(X)e-3'；
5'-(X)a-(NTarget)-(X)b-(Q)m-(X)c-(L)j-(X)d-(Z)h-(X)e-(P)k-(X)f-3'; or
5'-(X)a-(NTarget)-(X)b-(Q)m-(X)c-(L)j-(X)d-(Z)h-(X)e-(P)k-(X)f-(F)i-3'。
Here, NTargetIs a base sequence capable of forming a complementary bond with a target sequence on a target gene or nucleic acid, and is determined according to the target sequence on the target gene or nucleic acidA region of the base sequence to be altered.
(Q)mComprising a base sequence comprising a first complementary domain capable of forming a complementary binding with a second complementary domain. (Q)mMay be a sequence having partial or complete homology to a first complementary domain of a naturally occurring species; the base sequence of the first complementary domain may be altered depending on the species of origin. Q may each be independently selected from the group consisting of A, U, C and G; m may be the number of bases, which may be an integer of 5 to 35.
For example, when the first complementary domain has partial or complete homology with the first complementary domain of Streptococcus pyogenes or the first complementary domain derived from Streptococcus pyogenes, (Q)mMay be 5'-GUUUUAGAGCUA-3' or a base sequence having at least 50% or more homology to 5'-GUUUUAGAGCUA-3'.
In another example, when the first complementary domain has partial or complete homology with the first complementary domain of Campylobacter jejuni or the first complementary domain derived from Campylobacter jejuni, (Q)mMay be 5'-GUUUUAGUCCCUUUUUAAAUUUCUU-3' or 5'-GUUUUAGUCCCUU-3', or a base sequence having at least 50% or more homology to 5'-GUUUUAGUCCCUUUUUAAAUUUCUU-3' or 5'-GUUUUAGUCCCUU-3'.
In yet another example, (Q) when the first complementing domain has partial or complete homology with the first complementing domain of S.thermophilus or the first complementing domain derived from S.thermophilusmMay be 5'-GUUUUAGAGCUGUGUUGUUUCG-3' or a base sequence having at least 50% or more homology to 5'-GUUUUAGAGCUGUGUUGUUUCG-3'.
Furthermore, (L)jIs a base sequence comprising a linker domain that links the first complementary domain and the second complementary domain, thereby producing a single-stranded gRNA. Here, L may each be independently selected from the group consisting of A, U, C and G; j may be the number of bases, which is an integer from 1 to 30.
(Z)hIs a base sequence comprising a second complementary domain capable of forming a complementary binding with the first complementary domain. (Z)hCan be anda sequence having partial or complete homology to a second complementary domain of a naturally occurring species; the base sequence of the second complementary domain may be altered depending on the species of origin. Each Z may be independently selected from the group consisting of A, U, C and G; h is the number of bases, which can be an integer from 5 to 50.
For example, when the second complementary domain has partial or complete homology with a second complementary domain of Streptococcus pyogenes or a second complementary domain derived from Streptococcus pyogenes, (Z)hMay be 5'-UAGCAAGUUAAAAU-3' or a base sequence having at least 50% or more homology to 5'-UAGCAAGUUAAAAU-3'.
In another example, when the second complementary domain has partial or complete homology with the second complementary domain of Campylobacter jejuni or the second complementary domain derived from Campylobacter jejuni, (Z)hMay be 5'-AAGAAAUUUAAAAAGGGACUAAAAU-3' or 5'-AAGGGACUAAAAU-3', or a base sequence having at least 50% or more homology to 5'-AAGAAAUUUAAAAAGGGACUAAAAU-3' or 5'-AAGGGACUAAAAU-3'.
In yet another example, (Z) when the second complementing domain has partial or complete homology with the second complementing domain of S.thermophilus or the second complementing domain derived from S.thermophilushMay be 5'-CGAAACAACACAGCGAGUUAAAAU-3' or a base sequence having at least 50% or more homology to 5'-CGAAACAACACAGCGAGUUAAAAU-3'.
(P)kIs a base sequence comprising a proximal domain, which may have partial or complete homology with the proximal domain of a naturally occurring species; the base sequence of the proximal domain may be modified depending on the species of origin. Each P may be independently selected from the group consisting of A, U, C and G; k may be the number of bases, which is an integer of 1 to 20.
For example, when the proximal domain has partial or complete homology with the proximal domain of Streptococcus pyogenes or a proximal domain derived from Streptococcus pyogenes, (P)kMay be 5'-AAGGCUAGUCCG-3' or a base sequence having at least 50% or more homology to 5'-AAGGCUAGUCCG-3'.
In another example, when the proximal domain has partial or complete homology with the proximal domain of Campylobacter jejuni or the proximal domain derived from Campylobacter jejuni, (P)kMay be 5'-AAAGAGUUUGC-3' or a base sequence having at least 50% or more homology to 5'-AAAGAGUUUGC-3'.
In yet another example, when the proximal domain has partial or complete homology to the proximal domain of S.thermophilus or to a proximal domain derived from S.thermophilus, (P)kMay be 5'-AAGGCUUAGUCCG-3' or a base sequence having at least 50% or more homology to 5'-AAGGCUUAGUCCG-3'.
(F)iMay be a base sequence comprising a tail domain, which may have partial or complete homology with the tail domain of a naturally occurring species; the base sequence of the tail domain may be modified depending on the species of origin. Each F may be independently selected from the group consisting of A, U, C and G; i may be the number of bases, which is an integer of 1 to 50.
For example, when the tail domain has partial or complete homology with the tail domain of Streptococcus pyogenes or a tail domain derived from Streptococcus pyogenes, (F)iMay be 5'-UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3' or a base sequence having at least 50% or more homology to 5'-UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3'.
In another example, when the tail domain has partial or complete homology with the tail domain of Campylobacter jejuni or the tail domain derived from Campylobacter jejuni, (F)iMay be 5'-GGGACUCUGCGGGGUUACAAUCCCCUAAAACCGCUUUU-3' or a base sequence having at least 50% or more homology to 5'-GGGACUCUGCGGGGUUACAAUCCCCUAAAACCGCUUUU-3'.
In yet another example, (F) when the tail domain has partial or complete homology to the tail domain of S.thermophilus or a tail domain derived from S.thermophilusiMay be 5'-UACUCAACUUGAAAAGGUGGCACCGAUUCGGUGUUUUU-3' or a base sequence having at least 50% or more homology to 5'-UACUCAACUUGAAAAGGUGGCACCGAUUCGGUGUUUUU-3'.
Furthermore, (F)iThe 3' end may comprise a sequence of 1-10 bases involved in vitro or in vivo transcription methods.
For example, when the T7 promoter is used for in vitro transcription of grnas, the tail domain can be any base sequence present at the 3' end of the DNA template. Furthermore, when the U6 promoter is used for in vivo transcription, the tail domain may be UUUUUU; when the H1 promoter is used for transcription, the tail domain may be a uuuuuu; and when a pol-III promoter is used, the tail domain may contain several uracil bases or alternative bases.
Furthermore, (X)a、(X)b、(X)c、(X)d、(X)eAnd (X)fOptionally added base sequence, wherein each X can be independently selected from the group consisting of A, U, C and G; a. b, c, d, e and f may each be a number of bases which is 0 or an integer of 1 to 20.
Second Single-stranded gRNA
The second single-stranded gRNA may be a single-stranded gRNA consisting of a guide domain, a first complementary domain, and a second complementary domain.
Here, the second single-stranded gRNA may be composed of
5'- [ second complementary domain ] - [ first complementary domain ] - [ directing domain ] -3'; or
5'- [ second complementary domain ] - [ linker domain ] - [ first complementary domain ] - [ directing domain ] -3'.
The second single-stranded gRNA can optionally comprise additional nucleotide sequences.
In an exemplary embodiment, the second single-stranded gRNA may be
5'-(Z)h-(Q)m-(NTarget) -3'; or
5'-(X)a-(Z)h-(X)b-(Q)m-(X)c-(NTarget)-3'。
In another embodiment, the single-stranded gRNA can be a single-stranded gRNA
5'-(Z)h-(L)j-(Q)m-(NTarget) -3'; or
5'-(X)a-(Z)h-(L)j-(Q)m-(X)c-(NTarget)-3'。
Here, NTargetIs a base sequence capable of forming a complementary bond with a target sequence on a target gene or a nucleic acid, and is a base sequence region which can be changed depending on the target sequence on the target gene or the nucleic acid.
(Q)mIs a base sequence comprising a first complementary domain capable of forming a complementary binding with a second complementary domain of a second strand. (Q)mMay be a sequence having partial or complete homology to a first complementary domain of a naturally occurring species; the base sequence of the first complementary domain may be altered depending on the species of origin. Q may each be independently selected from the group consisting of A, U, C and G; m may be the number of bases, which may be an integer of 5 to 35.
For example, when the first complementary domain has partial or complete homology with the first complementary domain of the thrifty bacterium or the first complementary domain derived therefrom, (Q)mMay be 5 '-UUGUAGAU-3' or a base sequence having at least 50% or more homology to 5 '-UUGUAGAU-3'.
(Z)hIs a base sequence comprising a second complementary domain capable of forming a complementary binding with the first complementary domain of the first strand. (Z)hMay be a sequence having partial or complete homology to a second complementary domain of a naturally occurring species; the base sequence of the second complementary domain may be modified depending on the species of origin. Each Z may be independently selected from the group consisting of A, U, C and G; h may be the number of bases, which is an integer of 5 to 50.
For example, when the second complementary domain has partial or complete homology with the second complementary domain of the thrifty bacterium or the second complementary domain derived from the thrifty bacterium, (Z)hMay be 5'-AAAUUUCUACU-3' or a base sequence having at least 50% or more homology to 5'-AAAUUUCUACU-3'.
Furthermore, (L)jIs a base sequence comprising a linker domain that links a first complementary domain and a second complementary domain. Here, L may be each independentlySelected from the group consisting of A, U, C and G; j may be the number of bases, which is an integer from 1 to 30.
Furthermore, (X)a、(X)bAnd (X)cEach is an optional additional base sequence, wherein each X may be independently selected from the group consisting of A, U, C and G; a. b and c may be the number of bases, which is 0 or an integer of 1 to 20.
As aspects of the disclosure, the guide nucleic acid may be a gRNA capable of forming a complementary binding with a target sequence of an immunomodulatory gene.
The term "immunomodulatory genes" refers to all genes that are directly involved in or indirectly affect the regulation of immune function or the regulation of functions associated with the development and performance of an immune response. In the present invention, the immunoregulatory gene includes all genes directly involved in or indirectly affecting the regulation of the functions of immune cells, phagocytes and the like capable of interacting with immune cells. In particular, the immunomodulatory genes may perform immune functions or functions related to the development and performance of immune responses due to the immunomodulatory genes themselves or proteins expressed by the immunomodulatory genes.
Immunomodulatory genes can be classified according to the function of the protein expressed by the immunomodulatory gene. The immunomodulatory genes listed below are merely examples of function-based immunomodulatory genes and are not intended to limit the types of immunomodulatory genes encompassed by the invention. The genes listed below may have not only one type of immunomodulatory function, but may have multiple types of functions. In addition, more than two immunomodulatory genes may be provided (if desired).
In one example, the immune modulatory gene can be an immune cell activity modulating gene.
The term "immune cell activity-regulating gene" is a gene that functions to regulate the degree or activity of an immune response, and for example, it may be a gene that stimulates or suppresses the degree or activity of an immune response. Here, the immune cell activity-regulating gene may perform the following functions: the extent or activity of the immune response is controlled by the immune cell activity modulating gene or by a protein expressed by the immune cell activity modulating gene.
The immune cell activity-regulating gene may perform a function associated with activation or inactivation of an immune cell.
The immune cell activity-regulating gene may perform a function associated with activation or inactivation of an immune cell.
The immune cell activity regulating gene can play a role in suppressing immune response.
The immune cell activity-regulating gene can bind to a channel protein and a receptor of a cell membrane, thereby performing a function related to the synthesis of a protein regulating an immune response.
For example, the immune cell activity modulating gene may be a programmed cell death protein (PD-1)
The PD-1 gene (also referred to as PDCD1 gene; hereinafter the PD-1 gene and PDCD1 gene are used to represent the same gene) refers to a gene (full-length DNA, cDNA, or mRNA) encoding a PD-1 protein (also referred to as cluster of differentiation 279(CD 279)). In embodiments, the PD-1 gene may be one or more selected from the group consisting of, but not limited to: a gene encoding human PD-1 (e.g., NCBI accession No. NP-005009.2, etc.), for example, a PD-1 gene represented by NCBI accession No. NM-005018.2, NG-012110.1, etc.
The immune cell activity regulating gene may be cytotoxic T lymphocyte-associated protein 4 (CTLA-4).
The CTLA-4 gene refers to a gene (full-length DNA, cDNA or mRNA) encoding CTLA-4 protein (also referred to as cluster of differentiation 152(CD 152)). In embodiments, the CTLA-4 gene may be one or more selected from the group consisting of, but not limited to: genes encoding human CTLA-4 (e.g., NCBI accession numbers NP _001032720.1, NP _005205.2, etc.), such as CTLA-4 genes represented by NCBI accession numbers NM _001037631.2, NM _005214.4, NG _011502.1, etc.
The immune cell activity modulating gene may be CBLB.
The immune cell activity regulating gene may be PSGL-1.
The immune cell activity modulating gene may be ILT 2.
The immune cell activity modulating gene may be KIR2DL 4.
The immune cell activity modulating gene may be SHP-1.
The gene can be derived from mammals including primates (e.g., humans, monkeys, etc.), rodents (e.g., mice, rats, etc.).
Genetic information can be obtained from known databases, such as GenBank of the National Center for Biotechnology Information (NCBI).
In one embodiment, the immune cell activity modulating gene may act to stimulate an immune response.
The immune cell activity regulatory gene may be an immune cell growth regulatory gene.
The term "immune cell growth regulatory gene" refers to a gene that regulates the growth of an immune cell by regulating the synthesis of a protein in the immune cell or the like, for example, a gene that stimulates or inhibits the growth of an immune cell. In this case, the immune cell growth regulatory gene may perform a function of controlling the growth of the immune cell by controlling protein synthesis in the immune cell having the immune cell growth regulatory gene itself or a protein expressed by the immune cell growth regulatory gene.
Immune cell growth regulatory genes may function in DNA transcription, RNA translation, and cell differentiation.
Examples of immune cell growth regulatory genes may be genes involved in the expression pathways of NFAT, IkB/NF-kappa B, AP-1, 4E-BP1, eIF4E and S6.
For example, the immune cell growth regulatory gene can be DGK- α.
The DGKA (Dgk-alpha, DGK α) gene refers to a gene (full length DNA, cDNA or mRNA) encoding diacylglycerol kinase α protein (DGKA). in embodiments, the DGKA gene may be one or more selected from the group consisting of, but not limited to, a gene encoding human DGKA (e.g., NCBI accession numbers NP-001336.2, NP-958852.1, NP-958853.1, NP-963848.1, etc.), such as those represented by NCBI accession numbers NM-001345.4, NM-201444.2, NM-201445.1, NM-201554.1, NC-000012.12, etc.
The immune cell growth regulatory gene may be DGK-zeta.
The DGKZ (Dgk-zeta, DGK zeta) gene refers to a gene (full-length DNA, cDNA or mRNA) encoding diacylglycerol kinase zeta protein (DGKZ). In embodiments, the DGKZ gene may be one or more selected from the group consisting of, but not limited to: genes encoding human DGKZ (e.g., NCBI accession numbers NP _001099010.1, NP _001186195.1, NP _001186196.1, NP _001186197.1, NP _003637.2, NP _963290.1, NP _963291.2, etc.), such as DGKZ genes represented by NCBI accession numbers NM _001105540.1, NM _001199266.1, NM _001199267.1, NM _001199268.1, NM _003646.3, NM _201532.2, NM _201533.3, NG _047092.1, etc.
The immune cell growth regulatory gene may be EGR 2.
The EGR2 gene refers to the gene (full length DNA, cDNA or mRNA) encoding early growth response protein 2(EGR 2). In embodiments, EGR2 gene may be one or more selected from the group consisting of, but not limited to: genes encoding human EGR2 (e.g., NCBI accession numbers NP-000390, NP-001129649, NP-001129650, NP-001129651, NP-001307966, etc.). For example, an EGR2 gene represented by NCBI accession numbers NM _000399, NM _001136177, NM _001136178, NM _001136179, NM _001321037, and the like.
The immune cell growth regulatory gene may be EGR 3.
The immune cell growth regulatory gene may be PPP2r2 d.
The immune cell growth regulatory gene may be a20(TNFAIP 3).
The above genes can be derived from mammals including primates (e.g., humans, monkeys, etc.), rodents (e.g., mice, rats, etc.).
Genetic information can be obtained from known databases, such as GenBank of the National Center for Biotechnology Information (NCBI).
In embodiments, the immune cell activity modulating gene may be an immune cell death modulating gene.
The term "immune cell death regulatory gene" refers to a gene whose function is involved in immune cell death, e.g., stimulating or inhibiting the death of an immune cell. Here, the immune cell death regulatory gene may perform a function of controlling the death of the immune cell by the immune cell death regulatory gene itself or a protein expressed by the immune cell death regulatory gene.
The immune cell death regulatory gene may perform a function associated with apoptosis or necrosis of immune cells.
For example, the immune cell death regulatory gene may be a caspase cascade-associated gene (caspase-associated gene).
In this case, the immune cell death regulatory element can be Fas. When reference is made below to a gene, it will be apparent to one of ordinary skill in the art that the receptor or binding region on which the gene acts can be manipulated.
The immune cell death regulatory gene may be a death domain (death domain) -related gene.
Here, the immune cell death regulatory gene may be Daxx.
The immune cell death regulatory gene can be a Bcl-2 family gene.
The immune cell death regulatory gene can be a BH3-only family gene.
The immune cell death regulatory gene may be Bim.
The immune cell death regulatory gene can be Bid.
The immune cell death regulatory gene may be BAD.
The immune cell death regulatory gene may be a gene encoding a ligand or receptor located on the outer membrane of an immune cell.
Here, the immune cell death regulatory gene may be PD-1.
In addition, the immune cell death regulatory gene can be CTLA-4.
The above genes can be derived from mammals including primates (e.g., humans, monkeys, etc.), rodents (e.g., mice, rats, etc.).
Genetic information can be obtained from known databases, such as GenBank of the National Center for Biotechnology Information (NCBI).
In embodiments, the immune cell activity modulator gene may be an immune cell depletion modulator gene.
The term "immune cell depletion regulatory gene" is a gene that performs a function associated with the gradual loss of immune cell function, and here, the immune cell depletion regulatory gene may perform a function of controlling the gradual loss of immune cell function through the immune cell depletion regulatory gene itself or a protein expressed by the immune cell depletion regulatory gene.
The immune cell-depletion regulatory gene may function to assist transcription or translation of a gene involved in immune cell inactivation.
Here, the function of facilitating transcription may be a function of demethylating the corresponding gene.
In addition, genes involved in immune cell inactivation include immune cell activity-regulating genes.
For example, the immune cell depletion regulator gene may be TET 2.
The TET2 gene refers to a gene (full-length DNA, cDNA or mRNA) encoding TET2(Tet methylcytosine dioxygenase 2). In embodiments, the TET2 gene may be one or more genes encoding human TET2 (e.g., NCBI accession nos. NP _001120680.1, NP _060098.3, etc.) (e.g., TET2 gene represented by NCBI accession nos. NM _001127208.2, NM _017628.4, NG _028191.1, etc.), but is not limited thereto.
The immune cell-depleting regulatory elements may function to participate in the overgrowth of immune cells. Here, immune cells that undergo overgrowth and are not regenerated will lose their function.
Here, the immune cell depletion regulatory gene may be Wnt.
In addition, the immune cell depletion regulator gene can be Akt.
The above genes can be derived from mammals including primates (e.g., humans, monkeys, etc.), rodents (e.g., mice, rats, etc.).
Genetic information can be obtained from known databases, such as GenBank of the National Center for Biotechnology Information (NCBI).
In another embodiment, the regulatory element of immune cell activity can be a cytokine production regulatory gene.
The term "cytokine production regulatory gene" is an element involved in cytokine secretion of immune cells, which is expressed by immune cells performing such functions, and here, the cytokine production regulatory gene may exert a function of controlling cytokine production of immune cells by the cytokine production regulatory gene itself or a protein expressed by the cytokine production regulatory gene.
Cytokines are a general term for proteins secreted from immune cells, and are signal proteins that play important roles in the body. Cytokines are implicated in infection, immunity, inflammation, trauma, ulceration, cancer, and the like. Cytokines can be secreted by a cell and subsequently affect other cells, or affect cells that secrete themselves. For example, cytokines may induce macrophage proliferation or promote differentiation of secretory cells themselves. However, when the cytokine secretion is excessive, problems such as attack on normal cells may be caused, and thus proper secretion of the cytokine is also important in immune response.
The cytokine production regulatory gene may preferably be, for example, a gene in the secretion pathway of TNF α, IFN-. gamma.TGF- β, IL-2, IL-4, IL-10, IL-13, IL-1, IL-6, IL-12 and IFN- α.
Alternatively, cytokines may function to deliver signals to other immune cells to induce the immune cells to kill recognized antigen bearing cells or to aid in differentiation. In this case, the cytokine production regulatory gene may preferably be a gene in a gene pathway involved in IL-2 secretion.
The above genes can be derived from mammals including primates (e.g., humans, monkeys, etc.), rodents (e.g., mice, rats, etc.).
Genetic information can be obtained from known databases, such as GenBank of the National Center for Biotechnology Information (NCBI).
In embodiments, an immunomodulatory gene disclosed herein can be an immune cell activity modulating gene.
The immune regulatory gene can be PD-1 gene, CTLA-4 gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene, PPP2r2d gene, TET2 gene, PSGL-1 gene, A20 gene and/or KDM6A gene.
In one embodiment of the disclosure, the guide nucleic acid can be a gRNA that binds complementarily to a target sequence of the PD-1 gene, CTLA-4 gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene, PPP2r2d gene, TET2 gene, PSGL-1 gene, A20 gene, and/or KDM6A gene.
The term "target sequence" refers to a nucleotide sequence in a target gene or nucleic acid, particularly a partial nucleotide sequence of a target region in a target gene or nucleic acid, wherein a "target region" is a region in a target gene or nucleic acid that can be modified by a guide nucleic acid-editing protein.
The target genes disclosed herein can be immunomodulatory genes.
The target gene disclosed in the specification may be a PD-1 gene, CTLA-4 gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene, PPP2r2d gene, TET2 gene, PSGL-1 gene, A20 gene and/or KDM6A gene.
Hereinafter, the term "target sequence" may refer to two kinds of nucleotide sequence information. For example, for a target gene, the target sequence may refer to the sequence information of the transcribed strand of the target gene DNA, or the nucleotide sequence information of the non-transcribed strand.
For example, the target sequence may refer to a part of the nucleotide sequence (transcribed strand) 5'-ATCATTGGCAGACTAGTTCG-3' in the target region of the target gene a or the nucleotide sequence (non-transcribed strand) 5'-CGAACTAGTCTGCCAATGAT-3' complementary thereto.
The target sequence may be a sequence of 5-50 nucleotides.
In embodiments, the target sequence may be a nucleotide sequence of 16bp, 17bp, 18bp, 19bp, 20bp, 21bp, 22bp, 23bp, 24bp, or 25 bp.
The target sequence comprises a guide nucleic acid binding sequence or a guide nucleic acid non-binding sequence.
The term "guide nucleic acid binding sequence" refers to a nucleotide sequence having partial or complete complementarity to a guide sequence contained in the guide domain of a guide nucleic acid, which can form a complementary binding with a guide sequence contained in the guide domain of a guide nucleic acid. The target sequence and the guide nucleic acid binding sequence are nucleotide sequences that can be changed according to a target gene or nucleic acid (i.e., an object of gene manipulation or modification), and can be designed in various forms according to the target gene or nucleic acid.
The term "guide nucleic acid non-binding sequence" refers to a nucleotide sequence having partial or complete homology with a guide sequence contained in the guide domain of a guide nucleic acid, which is not capable of forming complementary binding with a guide sequence contained in the guide domain of a guide nucleic acid. In addition, the guide nucleic acid non-binding sequence is a nucleotide sequence complementary to the guide nucleic acid binding sequence, and can form complementary binding with the guide nucleic acid binding sequence.
The guide nucleic acid binding sequence is a partial nucleotide sequence in the target sequence, and may be either one of two nucleotide sequences having sequences different from the sequence of the target sequence, i.e., two nucleotide sequences that form complementary binding. Here, the guide nucleic acid non-binding sequence may be a nucleotide sequence of the target sequence other than the guide nucleic acid binding sequence.
For example, when the partial nucleotide sequence 5'-ATCATTGGCAGACTAGTTCG-3' and the nucleotide sequence 5'-CGAACTAGTCTGCCAATGAT-3' complementary thereto in the target region of the target gene a are target sequences, the guide nucleic acid binding sequence may be either of the two target sequences, i.e., 5'-ATCATTGGCAGACTAGTTCG-3' or 5'-CGAACTAGTCTGCCAATGAT-3'. Here, when the guide nucleic acid binding sequence is 5'-ATCATTGGCAGACTAGTTCG-3', the guide nucleic acid non-binding sequence may be 5'-CGAACTAGTCTGCCAATGAT-3'; or when the guide nucleic acid binding sequence is 5'-CGAACTAGTCTGCCAATGAT-3', the guide nucleic acid non-binding sequence can be 5'-ATCATTGGCAGACTAGTTCG-3'.
The guide nucleic acid binding sequence may be a nucleotide sequence selected from a nucleotide sequence homologous to the target sequence (i.e., the transcribed strand) and a nucleotide sequence homologous to the non-transcribed strand. Here, the guide nucleic acid non-binding sequence may be a nucleotide sequence selected from the group consisting of a nucleotide sequence homologous to the guide nucleic acid binding sequence (i.e., the transcribed strand) in the target sequence and a nucleotide sequence other than the nucleotide sequence homologous to the non-transcribed strand.
The guide nucleic acid binding sequence may be the same length as the target sequence.
The guide nucleic acid non-binding sequence may have the same length as the target sequence or the guide nucleic acid binding sequence.
The guide nucleic acid binding sequence may be a sequence of 5-50 nucleotides.
In embodiments, the guide nucleic acid binding sequence may be a nucleotide sequence of 16bp, 17bp, 18bp, 19bp, 20bp, 21bp, 22bp, 23bp, 24bp, or 25 bp.
The guide nucleic acid non-binding sequence may be a nucleotide sequence of 5bp to 50 bp.
In embodiments, the guide nucleic acid non-binding sequence may be a nucleotide sequence of 16bp, 17bp, 18bp, 19bp, 20bp, 21bp, 22bp, 23bp, 24bp, or 25 bp.
The guide nucleic acid binding sequence may form a partial or complete complementary binding with a guide sequence contained in a guide domain of the guide nucleic acid, and the length of the guide nucleic acid binding sequence may be the same as the length of the guide sequence.
The guide nucleic acid binding sequence can be a nucleotide sequence complementary to a guide sequence contained in a guide domain of the guide nucleic acid, having, for example, at least 70%, 75%, 80%, 85%, 90%, or 95% or more complementarity or complete complementarity.
In one example, the guide nucleic acid binding sequence may have or comprise a nucleotide sequence of 1bp to 8bp that is not complementary to a guide sequence comprised in the guide domain of the guide nucleic acid.
The guide nucleic acid non-binding sequence may have partial or complete homology with a guide sequence included in a guide domain of the guide nucleic acid, and the length of the guide nucleic acid non-binding sequence may be the same as the length of the guide sequence.
The guide nucleic acid non-binding sequence may be a nucleotide sequence having homology, for example, at least 70%, 75%, 80%, 85%, 90% or 95% or more homology or complete homology, with a guide sequence contained in a guide domain of the guide nucleic acid.
In one example, the guide nucleic acid non-binding sequence may have or comprise a nucleotide sequence of 1bp to 8bp that is not complementary to a guide sequence comprised in the guide domain of the guide nucleic acid.
The guide nucleic acid non-binding sequence may form a complementary binding with the guide nucleic acid binding sequence, and the length of the guide nucleic acid non-binding sequence may be the same as the length of the guide nucleic acid binding sequence.
The guide nucleic acid non-binding sequence may be a nucleotide sequence complementary to the guide nucleic acid binding sequence, having, for example, at least 90% or 95% or greater complementarity or complete complementarity.
In one example, the guide nucleic acid non-binding sequence may have or comprise a 1bp-2bp nucleotide sequence that is not complementary to the guide nucleic acid binding sequence.
In addition, the guide nucleic acid binding sequence may be a nucleotide sequence located near a nucleotide sequence that can be recognized by the editing protein.
In one example, the guide nucleic acid binding sequence may be a contiguous 5bp to 50bp nucleotide sequence adjacent to the 5 'end and/or 3' end of the nucleotide sequence capable of being recognized by the editing protein.
In embodiments, a target sequence disclosed herein can be a contiguous 10bp-35bp nucleotide sequence located in the promoter region of an immunomodulatory gene.
Here, the target sequence may be a nucleotide sequence of 10bp-35bp, 15bp-35bp, 20bp-35bp, 25bp-35bp, or 30bp-35 bp.
Alternatively, the target sequence may be a nucleotide sequence of 10bp-15bp, 15bp-20bp, 20bp-25bp, 25bp-30bp, or 30bp-35 bp.
In one example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence located in the promoter region of the PD-1 gene.
In another example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence located in the promoter region of the CTLA-4 gene.
In one example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence located in the promoter region of the a20 gene.
In another example, the target sequence can be a contiguous 10bp-25bp nucleotide sequence located in the promoter region of the DGKA gene.
In one example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence located in the promoter region of the DGKZ gene.
In another example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence located in the promoter region of the FAS gene.
In one example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence located in the promoter region of the EGR2 gene.
In another example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence located in the promoter region of the PPP2r2d gene.
In one example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence located in the promoter region of the TET2 gene.
In another example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence located in the promoter region of the PSGL-1 gene.
In another example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence located in the promoter region of the KDM6A gene.
The target sequence disclosed herein may be a contiguous 10bp-35bp nucleotide sequence located in an intron region of an immunomodulatory gene.
Here, the target sequence may be a nucleotide sequence of 10bp-35bp, 15bp-35bp, 20bp-35bp, 25bp-35bp, or 30bp-35 bp.
Alternatively, the target sequence may be a nucleotide sequence of 10bp-15bp, 15bp-20bp, 20bp-25bp, 25bp-30bp, or 30bp-35 bp.
In one example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence located in an intron region of the PD-1 gene.
In another example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence located in an intron region of the CTLA-4 gene.
In one example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence located in an intron region of the a20 gene.
In another example, the target sequence can be a contiguous 10bp-25bp nucleotide sequence located in an intron region of the DGKA gene.
In one example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence located in an intron region of the DGKZ gene.
In another example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence located in an intron region of the FAS gene.
In one example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence located in an intron region of the EGR2 gene.
In another example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence located in an intron region of the PPP2r2d gene.
In one example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence located in an intron region of the TET2 gene.
In another example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence located in an intron region of the PSGL-1 gene.
In yet another example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence located in an intron region of the KDM6A gene.
The target sequence disclosed in the present specification may be a continuous 10bp-35bp nucleotide sequence located in an exon region of an immune regulatory gene.
Here, the target sequence may be a nucleotide sequence of 10bp-35bp, 15bp-35bp, 20bp-35bp, 25bp-35bp, or 30bp-35 bp.
Alternatively, the target sequence may be a nucleotide sequence of 10bp-15bp, 15bp-20bp, 20bp-25bp, 25bp-30bp, or 30bp-35 bp.
In one example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence located in an exon region of the PD-1 gene.
In another example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence located in an exon region of a CTLA-4 gene.
In one example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence located in an exon region of the a20 gene.
In another example, the target sequence can be a contiguous 10bp-25bp nucleotide sequence located in an exon region of the DGKA gene.
In one example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence located in an exon region of the DGKZ gene.
In another example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence located in an exon region of the FAS gene.
In one example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence located in an exon region of the EGR2 gene.
In another example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence located in an exon region of the PPP2r2d gene.
In one example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence located in an exon region of the TET2 gene.
In another example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence located in an exon region of the PSGL-1 gene.
In yet another example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence located in an exon region of the KDM6A gene.
The target sequence disclosed in the present specification may be a continuous 10bp-35bp nucleotide sequence located in the enhancer region of an immunomodulatory gene.
Here, the target sequence may be a nucleotide sequence of 10bp-35bp, 15bp-35bp, 20bp-35bp, 25bp-35bp, or 30bp-35 bp.
Alternatively, the target sequence may be a nucleotide sequence of 10bp-15bp, 15bp-20bp, 20bp-25bp, 25bp-30bp, or 30bp-35 bp.
In one example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence located in the enhancer region of the PD-1 gene.
In another example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence located in the enhancer region of the CTLA-4 gene.
In one example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence located in the enhancer region of the a20 gene.
In another example, the target sequence can be a contiguous 10bp-25bp nucleotide sequence located in the enhancer region of the DGKA gene.
In one example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence located in the enhancer region of the DGKZ gene.
In another example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence located in the enhancer region of the FAS gene.
In one example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence located in the enhancer region of the EGR2 gene.
In another example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence located in the enhancer region of the PPP2r2d gene.
In one example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence located in the enhancer region of the TET2 gene.
In another example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence located in the enhancer region of the PSGL-1 gene.
In yet another example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence located in the enhancer region of the KDM6A gene.
The target sequence disclosed in the present specification may be a contiguous 10bp-35bp nucleotide sequence located in a coding region, a non-coding region, or a combination thereof of an immunomodulatory gene.
Here, the target sequence may be a nucleotide sequence of 10bp-35bp, 15bp-35bp, 20bp-35bp, 25bp-35bp, or 30bp-35 bp.
Alternatively, the target sequence may be a nucleotide sequence of 10bp-15bp, 15bp-20bp, 20bp-25bp, 25bp-30bp, or 30bp-35 bp.
In one example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence located in a coding region, a non-coding region, or a combination thereof of the PD-1 gene.
In another example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence located in a coding region, a non-coding region, or a combination thereof of the CTLA-4 gene.
In one example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence located in the coding region, non-coding region, or a combination thereof of the a20 gene.
In another example, the target sequence can be a contiguous 10bp-25bp nucleotide sequence located in the coding region, the non-coding region, or a combination thereof of the DGKA gene.
In one example, the target sequence can be a contiguous 10bp-25bp nucleotide sequence located in a coding region, a non-coding region, or a combination thereof of the DGKZ gene.
In another example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence located in the coding region, non-coding region, or a combination thereof of the FAS gene.
In one example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence located in a coding region, a non-coding region, or a combination thereof of the EGR2 gene.
In another example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence located in the coding region, non-coding region, or a combination thereof of the PPP2r2d gene.
In one example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence located in the coding region, non-coding region, or a combination thereof of the TET2 gene.
In another example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence located in a coding region, a non-coding region, or a combination thereof of the PSGL-1 gene.
In yet another example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence located in the coding region, non-coding region, or a combination thereof of the KDM6A gene.
The target sequence disclosed in the present specification may be a continuous 10bp-35bp nucleotide sequence located in a promoter region, an enhancer region, a 3'-UTR region, a 5' -UTR region, a polyA region, or a combination thereof, of an immune regulatory gene.
Here, the target sequence may be a nucleotide sequence of 10bp-35bp, 15bp-35bp, 20bp-35bp, 25bp-35bp, or 30bp-35 bp.
Alternatively, the target sequence may be a nucleotide sequence of 10bp-15bp, 15bp-20bp, 20bp-25bp, 25bp-30bp, or 30bp-35 bp.
In one example, the target sequence can be a contiguous 10bp-25bp nucleotide sequence located in a promoter region, enhancer region, 3'-UTR region, 5' -UTR region, polyA region, or a combination thereof, of the PD-1 gene.
In another example, the target sequence can be a contiguous 10bp-25bp nucleotide sequence located in a promoter region, enhancer region, 3'-UTR region, 5' -UTR region, polyA region, or a combination thereof, of the CTLA-4 gene.
In one example, the target sequence can be a contiguous 10bp-25bp nucleotide sequence located in the promoter region, enhancer region, 3'-UTR region, 5' -UTR region, polyA region, or a combination thereof, of the a20 gene.
In another example, the target sequence can be a contiguous 10bp-25bp nucleotide sequence located in a promoter region, enhancer region, 3'-UTR region, 5' -UTR region, polyA region, or a combination thereof, of the DGKA gene.
In one example, the target sequence can be a contiguous 10bp-25bp nucleotide sequence located in the promoter region, enhancer region, 3'-UTR region, 5' -UTR region, polyA region, or a combination thereof, of the DGKZ gene.
In another example, the target sequence can be a contiguous 10bp-25bp nucleotide sequence located in the promoter region, enhancer region, 3'-UTR region, 5' -UTR region, polyA region, or a combination thereof, of the FAS gene.
In one example, the target sequence can be a contiguous 10bp-25bp nucleotide sequence located in a promoter region, enhancer region, 3'-UTR region, 5' -UTR region, polyA region, or a combination thereof, of the EGR2 gene.
In another example, the target sequence can be a contiguous 10bp-25bp nucleotide sequence located in the promoter region, enhancer region, 3'-UTR region, 5' -UTR region, polyA region, or a combination thereof, of the PPP2r2d gene.
In one example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence located in the promoter region, enhancer region, 3'-UTR region, 5' -UTR region, polyA region, or a combination thereof, of the TET2 gene.
In another example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence located in a promoter region, enhancer region, 3'-UTR region, 5' -UTR region, polyA region, or a combination thereof, of the PSGL-1 gene.
In yet another example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence located in the promoter region, enhancer region, 3'-UTR region, 5' -UTR region, polyA region, or a combination thereof, of the KDM6A gene.
The target sequence disclosed in the present specification may be a continuous 10bp-35bp nucleotide sequence located in an exon region, an intron region, or a combination region of the immune regulatory gene.
Here, the target sequence may be a nucleotide sequence of 10bp-35bp, 15bp-35bp, 20bp-35bp, 25bp-35bp, or 30bp-35 bp.
Alternatively, the target sequence may be a nucleotide sequence of 10bp-15bp, 15bp-20bp, 20bp-25bp, 25bp-30bp, or 30bp-35 bp.
In one example, the target sequence can be a contiguous 10bp-25bp nucleotide sequence located in an exon region, an intron region, or a combination thereof, of the PD-1 gene.
In another example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence located in an exon region, an intron region, or a combination thereof, of the CTLA-4 gene.
In one example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence located in an exon region, an intron region, or a combination thereof, of the a20 gene.
In another example, the target sequence can be a contiguous 10bp-25bp nucleotide sequence located in an exon region, an intron region, or a combination thereof, of the DGKA gene.
In one example, the target sequence can be a contiguous 10bp-25bp nucleotide sequence located in an exon region, an intron region, or a combination thereof, of the DGKZ gene.
In another example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence located in an exon region, an intron region, or a combination thereof, of the FAS gene.
In one example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence located in an exon region, an intron region, or a combination thereof, of the EGR2 gene.
In another example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence located in an exon region, an intron region, or a combination thereof, of the PPP2r2d gene.
In one example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence located in an exon region, an intron region, or a combination thereof, of the TET2 gene.
In another example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence located in an exon region, an intron region, or a combination thereof, of the PSGL-1 gene.
In yet another example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence located in an exon region, an intron region, or a combination thereof, of the KDM6A gene.
The target sequence disclosed herein may be a contiguous 10bp-35bp nucleotide sequence comprising or adjacent to a mutated region of an immunomodulatory gene (e.g., a region different from the wild-type gene).
Here, the target sequence may be a nucleotide sequence of 10bp-35bp, 15bp-35bp, 20bp-35bp, 25bp-35bp, or 30bp-35 bp.
Alternatively, the target sequence may be a nucleotide sequence of 10bp-15bp, 15bp-20bp, 20bp-25bp, 25bp-30bp, or 30bp-35 bp.
In one example, the target sequence can be a contiguous 10bp-25bp nucleotide sequence comprising the PD-1 gene or a mutated region adjacent to the PD-1 gene (e.g., a region different from the wild-type gene).
In another example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence comprising or adjacent to a mutated region of a CTLA-4 gene (e.g., a region different from the wild-type gene).
In one example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence comprising or adjacent to a mutated region of the a20 gene (e.g., a region different from the wild-type gene).
In another example, the target sequence can be a contiguous 10bp-25bp nucleotide sequence comprising or adjacent to a mutant region of the DGKA gene (e.g., a region different from the wild-type gene).
In one example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence comprising or adjacent to a mutated region of the DGKZ gene (e.g., a region different from the wild-type gene).
In another example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence comprising or adjacent to a mutated region of the FAS gene (e.g., a region different from the wild-type gene).
In one example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence comprising or adjacent to a mutated region of EGR2 gene (e.g., a region different from the wild-type gene).
In another example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence comprising or adjacent to a mutated region of the PPP2r2d gene (e.g., a region different from the wild-type gene).
In one example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence comprising or adjacent to a mutated region of the TET2 gene (e.g., a region different from the wild-type gene).
In another example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence comprising or adjacent to a mutated region of the PSGL-1 gene (e.g., a region different from the wild-type gene).
In yet another example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence comprising or adjacent to a mutated region of the KDM6A gene (e.g., a region different from the wild-type gene).
The target sequence disclosed in the present specification may be a contiguous 10bp-35bp nucleotide sequence adjacent to the 5 'end and/or 3' end of a motif (PAM) sequence adjacent to a prepro-spacer sequence in a nucleic acid sequence of an immune regulatory gene.
The term "prepro-spacer sequence adjacent motif (PAM) sequence" is a nucleotide sequence that can be recognized by an edited protein. Here, the PAM sequence may have a nucleotide sequence that varies depending on the type and source species of the editing protein.
Here, the PAM sequence may be, for example, one or more of the following sequences (described in the 5 'to 3' direction):
NGG (N is A, T, C or G);
NNNNRYACs (each N is independently A, T, C or G; R is A or G; Y is C or T);
NNAGAAW (N is each independently A, T, C or G; W is A or T);
NNNNGATT (N is each independently A, T, C or G);
NNGRR (T) (N is A, T, C or G independently; R is A or G; Y is C or T); and
TTN (N is A, T, C or G).
Here, the target sequence may be a nucleotide sequence of 10bp-35bp, 15bp-35bp, 20bp-35bp, 25bp-35bp, or 30bp-35 bp.
Alternatively, the target sequence may be a nucleotide sequence of 10bp-15bp, 15bp-20bp, 20bp-25bp, 25bp-30bp, or 30bp-35 bp.
In one example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence adjacent to the 5 'end and/or 3' end of the PAM sequence in the nucleic acid sequence of the PD-1 gene.
In embodiments, when the PAM sequence recognized by the editing protein is 5'-NGG-3', 5'-NAG-3' or/and 5'-NGA-3' (N ═ A, T, G or C; or A, U, G or C), the target sequence may be a contiguous 10bp-25bp nucleotide sequence at the 5 'end or/and the 3' end of 5'-NGG-3', 5'-NAG-3' or/and 5'-NGA-3' (N ═ A, T, G or C; or A, U, G or C) in the nucleic acid sequence adjacent to the PD-1 gene.
In another embodiment, when the PAM sequence recognized by the editing protein is 5'-NGGNG-3' or/and 5 '-NNAGAAW-W-3' (W ═ a or T; N ═ A, T, G or C, or A, U, G or C), the target sequence may be a contiguous 10bp-25bp nucleotide sequence of the 5 'end or/and 3' end of 5'-NGGNG-3' or/and 5 '-NNAGAAW-W-3' (W ═ a or T; N ═ A, T, G or C, or A, U, G or C) in the nucleic acid sequence adjacent to the PD-1 gene.
In yet another embodiment, when the PAM sequence recognized by the editing protein is 5'-NNNNGATT-3' or/and 5'-NNNGCTT-3' (N ═ A, T, G or C; or A, U, G or C), the target sequence may be a contiguous 10bp-25bp nucleotide sequence of the 5 'end or/and 3' end of 5 '-nngatt-3' or/and 5'-NNNGCTT-3' (N ═ A, T, G or C; or A, U, G or C) in the nucleic acid sequence adjacent to the PD-1 gene.
In embodiments, when the PAM sequence recognized by the editing protein is 5'-NNNVRYAC-3' (V ═ G, C or a; R ═ a or G; Y ═ C or T; N ═ A, T, G or C, or A, U, G or C), the target sequence may be a contiguous 10bp-25bp nucleotide sequence of the 5'-NNNVRYAC-3' (V ═ G, C or a; R ═ a or G; Y ═ C or T; N ═ A, T, G or C, or A, U, G or C) at the 5 'end or/and the 3' end in the nucleic acid sequence adjacent to the PD-1 gene.
In another embodiment, when the PAM sequence recognized by the editing protein is 5'-NAAR-3' (R ═ a or G; N ═ A, T, G or C, or A, U, G or C), the target sequence may be a contiguous 10bp-25bp nucleotide sequence adjacent to the 5 'end or/and the 3' end of 5'-NAAR-3' (R ═ a or G; N ═ A, T, G or C, or A, U, G or C) in the nucleic acid sequence of the PD-1 gene.
In yet another embodiment, when the PAM sequence recognized by the editing protein is 5'-NNGRR-3', 5'-NNGRRT-3' or/and 5'-NNGRRV-3' (R ═ a or G; V ═ G, C or a; N ═ A, T, G or C, or A, U, G or C), the target sequence may be a contiguous 10bp-25bp nucleotide sequence of the 5 'end or 3' end of 5'-NNGRR-3', 5'-NNGRRT-3' or/and 5'-NNGRRV-3' (R ═ a or G; V ═ G, C or a; N ═ A, T, G or C, or A, U, G or C) in the nucleic acid sequence adjacent to the PD-1 gene.
In embodiments, when the PAM sequence recognized by the editing protein is 5'-TTN-3' (N ═ A, T, G or C; or A, U, G or C), the target sequence may be a contiguous 10-25 nucleotide sequence adjacent to the 5 'end or/and the 3' end of 5'-TTN-3' (N ═ A, T, G or C; or A, U, G or C) in the nucleic acid sequence of the PD-1 gene.
In another example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence adjacent to the 5 'end and/or 3' end of the PAM sequence in the nucleic acid sequence of the CTLA-4 gene.
In embodiments, when the PAM sequence recognized by the editing protein is 5'-NGG-3', 5'-NAG-3' or/and 5'-NGA-3' (N ═ A, T, G or C; or A, U, G or C), the target sequence may be a contiguous 10bp-25bp nucleotide sequence at the 5 'end or/and the 3' end of 5'-NGG-3', 5'-NAG-3' or/and 5'-NGA-3' (N ═ A, T, G or C; or A, U, G or C) in the nucleic acid sequence adjacent to the CTLA-4 gene.
In another embodiment, when the PAM sequence recognized by the editing protein is 5' -NGGNG-3' or/and 5' -NNAGAAW-W-3 ' (W ═ a or T; N ═ A, T, G or C, or A, U, G or C), the target sequence may be a contiguous 10bp-25bp nucleotide sequence of the 5' -NGGNG-3' or/and 5' -NNAGAAW-W-3 ' (W ═ a or T; N ═ A, T, G or C, or A, U, G or C) end or/and 3' end in the nucleic acid sequence adjacent to the CTLA-4 gene.
In yet another embodiment, when the PAM sequence recognized by the editing protein is 5'-NNNNGATT-3' or/and 5'-NNNGCTT-3' (N ═ A, T, G or C; or A, U, G or C), the target sequence may be a contiguous 10bp-25bp nucleotide sequence of the 5 'end or/and 3' end of 5 '-nngatt-3' or/and 5'-NNNGCTT-3' (N ═ A, T, G or C; or A, U, G or C) in the nucleic acid sequence adjacent to the CTLA-4 gene.
In embodiments, when the PAM sequence recognized by the editing protein is 5'-NNNVRYAC-3' (V ═ G, C or a; R ═ a or G; Y ═ C or T; N ═ A, T, G or C, or A, U, G or C), the target sequence may be a contiguous 10bp-25bp nucleotide sequence of the 5'-NNNVRYAC-3' (V ═ G, C or a; R ═ a or G; Y ═ C or T; N ═ A, T, G or C, or A, U, G or C) at the 5 'end or/and the 3' end in the nucleic acid sequence adjacent to the CTLA-4 gene.
In another embodiment, when the PAM sequence recognized by the editing protein is 5'-NAAR-3' (R ═ a or G; N ═ A, T, G or C, or A, U, G or C), the target sequence may be a contiguous 10bp-25bp nucleotide sequence adjacent to the 5 'end or/and the 3' end of 5'-NAAR-3' (R ═ a or G; N ═ A, T, G or C, or A, U, G or C) in the nucleic acid sequence of the CTLA-4 gene.
In yet another embodiment, when the PAM sequence recognized by the editing protein is 5'-NNGRR-3', 5'-NNGRRT-3' or/and 5'-NNGRRV-3' (R ═ a or G; V ═ G, C or a; N ═ A, T, G or C, or A, U, G or C), the target sequence may be a contiguous 10bp-25bp nucleotide sequence of the 5 'end or/and 3' end of 5'-NNGRR-3', 5'-NNGRRT-3' or/and 5'-NNGRRV-3' (R ═ a or G; V ═ G, C or a; N ═ A, T, G or C, or A, U, G or C) in the nucleic acid sequence adjacent to the CTLA-4 gene.
In embodiments, when the PAM sequence recognized by the editing protein is 5'-TTN-3' (N ═ A, T, G or C; or A, U, G or C), the target sequence may be a contiguous 10bp-25bp nucleotide sequence adjacent to the 5 'end or/and the 3' end of 5'-TTN-3' (N ═ A, T, G or C; or A, U, G or C) in the nucleic acid sequence of the CTLA-4 gene.
In one example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence adjacent to the 5 'end and/or 3' end of the PAM sequence in the nucleic acid sequence of the a20 gene.
In embodiments, when the PAM sequence recognized by the editing protein is 5'-NGG-3', 5'-NAG-3' or/and 5'-NGA-3' (N ═ A, T, G or C; or A, U, G or C), the target sequence may be a contiguous 10bp-25bp nucleotide sequence at the 5 'end or/and the 3' end of 5'-NGG-3', 5'-NAG-3' or/and 5'-NGA-3' (N ═ A, T, G or C; or A, U, G or C) in the nucleic acid sequence adjacent to the a20 gene.
In another embodiment, when the PAM sequence recognized by the editing protein is 5'-NGGNG-3' or/and 5'-NNAGAAW-3' (W ═ a or T; N ═ A, T, G or C, or A, U, G or C), the target sequence may be a contiguous 10bp-25bp nucleotide sequence of the 5 'end or/and 3' end of 5'-NGGNG-3' or/and 5'-NNAGAAW-3' (W ═ a or T; N ═ A, T, G or C, or A, U, G or C) in the nucleic acid sequence adjacent to the a20 gene.
In yet another embodiment, when the PAM sequence recognized by the editing protein is 5' -NNNNGATT-3' or/and 5' -NNNGCTT-3' (N ═ A, T, G or C; or A, U, G or C), the target sequence may be a contiguous 10bp-25bp nucleotide sequence of the 5' -nngatt-3 ' or/and 5' -NNNGCTT-3' (N ═ A, T, G or C; or A, U, G or C) end or/and 3' end in the nucleic acid sequence adjacent to the a20 gene.
In embodiments, when the PAM sequence recognized by the editing protein is 5'-NNNVRYAC-3' (V ═ G, C or a; R ═ a or G; Y ═ C or T; N ═ A, T, G or C, or A, U, G or C), the target sequence may be a contiguous 10bp-25bp nucleotide sequence of the 5'-NNNVRYAC-3' (V ═ G, C or a; R ═ a or G; Y ═ C or T; N ═ A, T, G or C, or A, U, G or C) at the 5 'end or/and the 3' end of the nucleic acid sequence adjacent to the a20 gene.
In another embodiment, when the PAM sequence recognized by the editing protein is 5'-NAAR-3' (R ═ a or G; N ═ A, T, G or C, or A, U, G or C), the target sequence may be a contiguous 10bp-25bp nucleotide sequence adjacent to the 5 'end or/and the 3' end of 5'-NAAR-3' (R ═ a or G; N ═ A, T, G or C, or A, U, G or C) in the nucleic acid sequence of the a20 gene.
In yet another embodiment, when the PAM sequence recognized by the editing protein is 5'-NNGRR-3', 5'-NNGRRT-3' or/and 5'-NNGRRV-3' (R ═ a or G; V ═ G, C or a; N ═ A, T, G or C, or A, U, G or C), the target sequence may be a contiguous 10bp-25bp nucleotide sequence of the 5 'end or/and 3' end of 5'-NNGRR-3', 5'-NNGRRT-3' or/and 5'-NNGRRV-3' (R ═ a or G; V ═ G, C or a; N ═ A, T, G or C, or A, U, G or C) in the nucleic acid sequence adjacent to the a20 gene.
In embodiments, when the PAM sequence recognized by the editing protein is 5'-TTN-3' (N ═ A, T, G or C; or A, U, G or C), the target sequence may be a contiguous 10bp-25bp nucleotide sequence adjacent to the 5 'end or/and the 3' end of 5'-TTN-3' (N ═ A, T, G or C; or A, U, G or C) in the nucleic acid sequence of the a20 gene.
In another example, the target sequence can be a contiguous 10bp-25bp nucleotide sequence adjacent to the 5 'end and/or 3' end of a PAM sequence in the nucleic acid sequence of the DGKA gene.
In embodiments, when the PAM sequence recognized by the editing protein is 5'-NGG-3', 5'-NAG-3' or/and 5'-NGA-3' (N ═ A, T, G or C; or A, U, G or C), the target sequence may be a contiguous 10bp-25bp nucleotide sequence at the 5 'end or/and the 3' end of 5'-NGG-3', 5'-NAG-3' or/and 5'-NGA-3' (N ═ A, T, G or C; or A, U, G or C) in the nucleic acid sequence adjacent to the DGKA gene.
In another embodiment, when the PAM sequence recognized by the editing protein is 5'-NGGNG-3' or/and 5'-NNAGAAW-3' (W ═ a or T; N ═ A, T, G or C, or A, U, G or C), the target sequence may be a contiguous 10bp to 25bp nucleotide sequence at the 5 'end or/and the 3' end of 5'-NGGNG-3' or/and 5'-NNAGAAW-3' (W ═ a or T; N ═ A, T, G or C, or A, U, G or C) in the nucleic acid sequence adjacent to the DGKA gene.
In yet another embodiment, when the PAM sequence recognized by the editing protein is 5' -NNNNGATT-3' or/and 5' -NNNGCTT-3' (N ═ A, T, G or C; or a, U, G or C), the target sequence may be a contiguous 10bp-25bp nucleotide sequence of the 5' -nngatt-3 ' or/and 5' -NNNGCTT-3' (N ═ A, T, G or C; or A, U, G or C) end or/and 3' end in the nucleic acid sequence adjacent to the DGKA gene.
In embodiments, when the PAM sequence recognized by the editing protein is 5'-NNNVRYAC-3' (V ═ G, C or a; R ═ a or G; Y ═ C or T; N ═ A, T, G or C, or A, U, G or C), the target sequence may be a contiguous 10bp-25bp nucleotide sequence of the 5'-NNNVRYAC-3' (V ═ G, C or a; R ═ a or G; Y ═ C or T; N ═ A, T, G or C, or A, U, G or C) at the 5 'end or/and the 3' end in the nucleic acid sequence adjacent to the DGKA gene.
In another embodiment, when the PAM sequence recognized by the editing protein is 5'-NAAR-3' (R ═ a or G; N ═ A, T, G or C, or A, U, G or C), the target sequence may be a contiguous 10bp-25bp nucleotide sequence at the 5 'end or/and the 3' end of 5'-NAAR-3' (R ═ a or G; N ═ A, T, G or C, or A, U, G or C) in the nucleic acid sequence adjacent to the DGKA gene.
In yet another embodiment, when the PAM sequence recognized by the editing protein is 5'-NNGRR-3', 5'-NNGRRT-3' or/and 5'-NNGRRV-3' (R ═ a or G; V ═ G, C or a; N ═ A, T, G or C, or A, U, G or C), the target sequence may be a contiguous 10bp-25bp nucleotide sequence of the 5 'end or/and 3' end of 5'-NNGRR-3', 5'-NNGRRT-3' or/and 5'-NNGRRV-3' (R ═ a or G; V ═ G, C or a; N ═ A, T, G or C, or A, U, G or C) in the nucleic acid sequence adjacent to the DGKA gene.
In one embodiment, when the PAM sequence recognized by the editing protein is 5'-TTN-3' (N ═ A, T, G or C; or A, U, G or C), the target sequence may be a contiguous 10bp-25bp nucleotide sequence at the 5 'end or/and the 3' end of 5'-TTN-3' (N ═ A, T, G or C; or A, U, G or C) in the nucleic acid sequence adjacent to the DGKA gene.
In one example, the target sequence may be a contiguous nucleotide sequence of 10bp to 25bp adjacent to the 5 'end or/and 3' end of the PAM sequence in the nucleic acid sequence of the DGKZ gene.
In embodiments, when the PAM sequence recognized by the editing protein is 5'-NGG-3', 5'-NAG-3' or/and 5'-NGA-3' (N ═ A, T, G or C; or A, U, G or C), the target sequence may be a contiguous 10bp-25bp nucleotide sequence at the 5 'end or/and the 3' end of 5'-NGG-3', 5'-NAG-3' or/and 5'-NGA-3' (N ═ A, T, G or C; or A, U, G or C) in the nucleic acid sequence adjacent to the DGKZ gene.
In another embodiment, when the PAM sequence recognized by the editing protein is 5'-NGGNG-3' or/and 5 '-NNAGAAW-W-3' (W ═ a or T; N ═ A, T, G or C, or A, U, G or C), the target sequence may be a contiguous 10bp-25bp nucleotide sequence of the 5 'end or/and 3' end of 5'-NGGNG-3' or/and 5 '-NNAGAAW-W-3' (W ═ a or T; N ═ A, T, G or C, or A, U, G or C) in the nucleic acid sequence adjacent to the DGKZ gene.
In yet another embodiment, when the PAM sequence recognized by the editing protein is 5'-NNNNGATT-3' or/and 5'-NNNGCTT-3' (N ═ A, T, G or C; or a, U, G or C), the target sequence may be a contiguous 10bp-25bp nucleotide sequence of the 5 'end or/and 3' end of 5 '-nngatt-3' or/and 5'-NNNGCTT-3' (N ═ A, T, G or C; or A, U, G or C) in the nucleic acid sequence adjacent to the DGKZ gene.
In embodiments, when the PAM sequence recognized by the editing protein is 5'-NNNVRYAC-3' (V ═ G, C or a; R ═ a or G; Y ═ C or T; N ═ A, T, G or C, or A, U, G or C), the target sequence may be a contiguous 10bp-25bp nucleotide sequence of the 5'-NNNVRYAC-3' (V ═ G, C or a; R ═ a or G; Y ═ C or T; N ═ A, T, G or C, or A, U, G or C) at the 5 'end or/and the 3' end in the nucleic acid sequence adjacent to the DGKZ gene.
In another embodiment, when the PAM sequence recognized by the editing protein is 5'-NAAR-3' (R ═ a or G; N ═ A, T, G or C, or A, U, G or C), the target sequence may be a contiguous 10bp-25bp nucleotide sequence adjacent to the 5 'end or/and the 3' end of 5'-NAAR-3' (R ═ a or G; N ═ A, T, G or C, or A, U, G or C) in the nucleic acid sequence of the DGKZ gene.
In yet another embodiment, when the PAM sequence recognized by the editing protein is 5'-NNGRR-3', 5'-NNGRRT-3' or/and 5'-NNGRRV-3' (R ═ a or G; V ═ G, C or a; N ═ A, T, G or C, or A, U, G or C), the target sequence may be a contiguous 10bp-25bp nucleotide sequence of the 5 'end or/and 3' end of 5'-NNGRR-3', 5'-NNGRRT-3' or/and 5'-NNGRRV-3' (R ═ a or G; V ═ G, C or a; N ═ A, T, G or C, or A, U, G or C) in the nucleic acid sequence adjacent to the DGKZ gene.
In embodiments, when the PAM sequence recognized by the editing protein is 5'-TTN-3' (N ═ A, T, G or C; or A, U, G or C), the target sequence may be a contiguous 10bp-25bp nucleotide sequence at the 5 'end or/and the 3' end of 5'-TTN-3' (N ═ A, T, G or C; or A, U, G or C) in the nucleic acid sequence adjacent to the DGKZ gene.
In another example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence adjacent to the 5 'end and/or 3' end of the PAM sequence in the nucleotide sequence of the FAS gene.
In embodiments, when the PAM sequence recognized by the editing protein is 5'-NGG-3', 5'-NAG-3' or/and 5'-NGA-3' (N ═ A, T, G or C; or A, U, G or C), the target sequence may be a contiguous 10bp-25bp nucleotide sequence at the 5 'end or/and the 3' end of 5'-NGG-3', 5'-NAG-3' or/and 5'-NGA-3' (N ═ A, T, G or C; or A, U, G or C) in the nucleic acid sequence adjacent to the FAS gene.
In another embodiment, when the PAM sequence recognized by the editing protein is 5'-NGGNG-3' or/and 5'-NNAGAAW-3' (W ═ a or T; N ═ A, T, G or C, or A, U, G or C), the target sequence may be a contiguous 10bp-25bp nucleotide sequence at the 5 'end or/and the 3' end of 5'-NGGNG-3' or/and 5'-NNAGAAW-3' (W ═ a or T; N ═ A, T, G or C, or A, U, G or C) in the nucleic acid sequence adjacent to the FAS gene.
In yet another embodiment, when the PAM sequence recognized by the editing protein is 5'-NNNNGATT-3' or/and 5'-NNNGCTT-3' (N ═ A, T, G or C; or A, U, G or C), the target sequence may be a contiguous 10bp-25bp nucleotide sequence of the 5 'end or/and 3' end of 5 '-nnnggatt-3' or/and 5'-NNNGCTT-3' (N ═ A, T, G or C; or A, U, G or C) in the nucleic acid sequence adjacent to the FAS gene.
In embodiments, when the PAM sequence recognized by the editing protein is 5'-NNNVRYAC-3' (V ═ G, C or a; R ═ a or G; Y ═ C or T; N ═ A, T, G or C, or A, U, G or C), the target sequence may be a contiguous 10bp-25bp nucleotide sequence adjacent to the 5 'end or/and the 3' end of 5'-NNNVRYAC-3' (V ═ G, C or a; R ═ a or G; Y ═ C or T; N ═ A, T, G or C, or A, U, G or C) in the nucleic acid sequence of the FAS gene.
In another embodiment, when the PAM sequence recognized by the editing protein is 5'-NAAR-3' (R ═ a or G; N ═ A, T, G or C, or A, U, G or C), the target sequence may be a contiguous 10bp-25bp nucleotide sequence adjacent to the 5 'end or/and the 3' end of 5'-NAAR-3' (R ═ a or G; N ═ A, T, G or C, or A, U, G or C) in the nucleic acid sequence of the FAS gene.
In yet another embodiment, when the PAM sequence recognized by the editing protein is 5'-NNGRR-3', 5'-NNGRRT-3' or/and 5'-NNGRRV-3' (R ═ a or G; V ═ G, C or a; N ═ A, T, G or C, or A, U, G or C), the target sequence may be a contiguous 10bp-25bp nucleotide sequence of the 5 'end or/and 3' end of 5'-NNGRR-3', 5'-NNGRRT-3' or/and 5'-NNGRRV-3' (R ═ a or G; V ═ G, C or a; N ═ A, T, G or C, or A, U, G or C) in the nucleic acid sequence adjacent to the FAS gene.
In embodiments, when the PAM sequence recognized by the editing protein is 5'-TTN-3' (N ═ A, T, G or C; or A, U, G or C), the target sequence may be a contiguous 10bp-25bp nucleotide sequence adjacent to the 5 'end or/and the 3' end of 5'-TTN-3' (N ═ A, T, G or C; or A, U, G or C) in the nucleic acid sequence of the FAS gene.
In one example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence adjacent to the 5 'end and/or 3' end of a promiscuous sequence adjacent motif (PAM) in a nucleic acid sequence of EGR2 gene.
In embodiments, when the PAM sequence recognized by the editing protein is 5'-NGG-3', 5'-NAG-3' or/and 5'-NGA-3' (N ═ A, T, G or C; or A, U, G or C), the target sequence may be a contiguous 10bp-25bp nucleotide sequence at the 5 'end or/and the 3' end of 5'-NGG-3', 5'-NAG-3' or/and 5'-NGA-3' (N ═ A, T, G or C; or A, U, G or C) in the nucleic acid sequence adjacent to the EGR2 gene.
In another embodiment, when the PAM sequence recognized by the editing protein is 5'-NGGNG-3' or/and 5'-NNAGAAW-3' (W ═ a or T; N ═ a, T, G or C, or A, U, G or C), the target sequence may be a contiguous nucleotide sequence of 10bp to 25bp at the 5 'end or/and 3' end of 5'-NGGNG-3' or/and 5'-NNAGAAW-3' (W ═ a or T; N ═ A, T, G or C, or A, U, G or C) in the nucleic acid sequence adjacent to the EGR2 gene.
In yet another embodiment, when the PAM sequence recognized by the editing protein is 5'-NNNNGATT-3' or/and 5'-NNNGCTT-3' (N ═ A, T, G or C; or A, U, G or C), the target sequence may be a contiguous 10bp-25bp nucleotide sequence of the 5 'end or/and 3' end of 5 '-nngatt-3' or/and 5'-NNNGCTT-3' (N ═ A, T, G or C; or A, U, G or C) in the nucleic acid sequence adjacent to the EGR2 gene.
In embodiments, when the PAM sequence recognized by the editing protein is 5'-NNNVRYAC-3' (V ═ G, C or a; R ═ a or G; Y ═ C or T; N ═ A, T, G or C, or A, U, G or C), the target sequence may be a contiguous 10bp-25bp nucleotide sequence of the 5'-NNNVRYAC-3' (V ═ G, C or a; R ═ a or G; Y ═ C or T; N ═ A, T, G or C, or A, U, G or C) or the 5 'end or/and the 3' end in the nucleic acid sequence adjacent to the EGR2 gene.
In another embodiment, when the PAM sequence recognized by the editing protein is 5'-NAAR-3' (R ═ a or G; N ═ A, T, G or C, or A, U, G or C), the target sequence may be a contiguous 10bp-25bp nucleotide sequence adjacent to the 5 'end or/and the 3' end of 5'-NAAR-3' (R ═ a or G; N ═ A, T, G or C, or A, U, G or C) in the nucleic acid sequence of the EGR2 gene.
In yet another embodiment, when the PAM sequence recognized by the editing protein is 5'-NNGRR-3', 5'-NNGRRT-3' or/and 5'-NNGRRV-3' (R ═ a or G; V ═ G, C or a; N ═ A, T, G or C, or A, U, G or C), the target sequence may be a contiguous 10bp-25bp nucleotide sequence of the 5 'end or/and 3' end of 5'-NNGRR-3', 5'-NNGRRT-3' or/and 5'-NNGRRV-3' (R ═ a or G; V ═ G, C or a; N ═ A, T, G or C, or A, U, G or C) in the nucleic acid sequence adjacent to EGR2 gene.
In embodiments, when the PAM sequence recognized by the editing protein is 5'-TTN-3' (N ═ A, T, G or C; or A, U, G or C), the target sequence may be a contiguous 10bp-25bp nucleotide sequence adjacent to the 5 'end or/and the 3' end of 5'-TTN-3' (N ═ A, T, G or C; or A, U, G or C) in the nucleic acid sequence of the EGR2 gene.
In another example, the target sequence may be a continuous 10bp-25bp nucleotide sequence adjacent to the 5 'end or/and 3' end of the PAM sequence in the nucleotide sequence of the PPP2r2d gene.
In embodiments, when the PAM sequence recognized by the editing protein is 5'-NGG-3', 5'-NAG-3' or/and 5'-NGA-3' (N ═ A, T, G or C; or A, U, G or C), the target sequence may be a contiguous 10bp-25bp nucleotide sequence of the 5 'end or/and the 3' end of 5'-NGG-3', 5'-NAG-3' or/and 5'-NGA-3' (N ═ A, T, G or C; or A, U, G or C) in the nucleic acid sequence adjacent to the PPP2r2d gene.
In another embodiment, when the PAM sequence recognized by the editing protein is 5'-NGGNG-3' or/and 5 '-NNAGAAW-W-3' (W ═ a or T; N ═ A, T, G or C, or A, U, G or C), the target sequence may be a contiguous 10bp-25bp nucleotide sequence at the 5 'end or/and the 3' end of 5'-NGGNG-3' or/and 5 '-NNAGAAW-W-3' (W ═ a or T; N ═ A, T, G or C, or A, U, G or C) in the nucleic acid sequence adjacent to the PPP2r2d gene.
In yet another embodiment, when the PAM sequence recognized by the editing protein is 5' -NNNNGATT-3' or/and 5' -NNNGCTT-3' (N ═ A, T, G or C; or A, U, G or C), the target sequence may be a contiguous 10bp-25bp nucleotide sequence of the 5' -nngatt-3 ' or/and 5' -NNNGCTT-3' (N ═ A, T, G or C; or A, U, G or C) end or/and 3' end in the nucleic acid sequence adjacent to the PPP2r2d gene.
In embodiments, when the PAM sequence recognized by the editing protein is 5'-NNNVRYAC-3' (V ═ G, C or a; R ═ a or G; Y ═ C or T; N ═ A, T, G or C, or A, U, G or C), the target sequence may be a contiguous 10bp-25bp nucleotide sequence adjacent to the 5 'end or/and the 3' end of 5'-NNNVRYAC-3' (V ═ G, C or a; R ═ a or G; Y ═ C or T; N ═ A, T, G or C, or A, U, G or C) in the nucleic acid sequence of the PPP2R2d gene.
In another embodiment, when the PAM sequence recognized by the editing protein is 5'-NAAR-3' (R ═ a or G; N ═ A, T, G or C, or A, U, G or C), the target sequence may be a contiguous 10bp-25bp nucleotide sequence adjacent to the 5 'end or/and the 3' end of 5'-NAAR-3' (R ═ a or G; N ═ A, T, G or C, or A, U, G or C) in the nucleic acid sequence of the PPP2R2d gene.
In a further embodiment, when the PAM sequence recognized by the editing protein is 5' -NNGRR-3', 5' -NNGRRT-3' or/and 5' -NNGRRV-3' (R ═ a or G; V ═ G, C or a; N ═ A, T, G or C, or A, U, G or C), the target sequence may be a contiguous 10bp-25bp nucleotide sequence of the 5' -NNGRR-3', 5' -NNGRRT-3' or/and 5' -grrv-3 ' (R ═ a or G; V ═ G, C or a; N ═ A, T, G or C, or A, U, G or C) end or/and the 3' end of the nucleic acid sequence adjacent to the PPP2R2d gene.
In embodiments, when the PAM sequence recognized by the editing protein is 5'-TTN-3' (N ═ A, T, G or C; or A, U, G or C), the target sequence may be a contiguous 10bp-25bp nucleotide sequence adjacent to the 5 'end or/and the 3' end of 5'-TTN-3' (N ═ A, T, G or C; or A, U, G or C) in the nucleic acid sequence of PPP2r2d gene.
In one example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence adjacent to the 5 'end or/and 3' end of the PAM sequence in the nucleic acid sequence of the TET2 gene.
In embodiments, when the PAM sequence recognized by the editing protein is 5'-NGG-3', 5'-NAG-3' or/and 5'-NGA-3' (N ═ A, T, G or C; or A, U, G or C), the target sequence may be a contiguous 10bp-25bp nucleotide sequence at the 5 'end or/and the 3' end of 5'-NGG-3', 5'-NAG-3' or/and 5'-NGA-3' (N ═ A, T, G or C; or A, U, G or C) in the nucleic acid sequence adjacent to the TET2 gene.
In another embodiment, when the PAM sequence recognized by the editing protein is 5' -NGGNG-3' or/and 5' -NNAGAAW-W-3 ' (W ═ a or T; N ═ A, T, G or C, or A, U, G or C), the target sequence may be a contiguous 10bp-25bp nucleotide sequence of the 5' -NGGNG-3' or/and 5' -NNAGAAW-W-3 ' (W ═ a or T; N ═ A, T, G or C, or A, U, G or C) or/and the 3' end of the nucleic acid sequence adjacent to the TET2 gene.
In yet another embodiment, when the PAM sequence recognized by the editing protein is 5' -NNNNGATT-3' or/and 5' -NNNGCTT-3' (N ═ A, T, G or C; or A, U, G or C), the target sequence may be a contiguous 10bp-25bp nucleotide sequence of the 5' -nngatt-3 ' or/and 5' -NNNGCTT-3' (N ═ A, T, G or C; or A, U, G or C) end or/and 3' end in the nucleic acid sequence adjacent to the TET2 gene.
In embodiments, when the PAM sequence recognized by the editing protein is 5'-NNNVRYAC-3' (V ═ G, C or a; R ═ a or G; Y ═ C or T; N ═ A, T, G or C, or A, U, G or C), the target sequence may be a contiguous 10bp-25bp nucleotide sequence of the 5'-NNNVRYAC-3' (V ═ G, C or a; R ═ a or G; Y ═ C or T; N ═ A, T, G or C, or A, U, G or C) or the 5 'end or/and the 3' end in the nucleic acid sequence adjacent to the TET2 gene.
In another embodiment, when the PAM sequence recognized by the editing protein is 5'-NAAR-3' (R ═ a or G; N ═ A, T, G or C, or A, U, G or C), the target sequence may be a contiguous 10bp-25bp nucleotide sequence adjacent to the 5 'end or/and the 3' end of 5'-NAAR-3' (R ═ a or G; N ═ A, T, G or C, or A, U, G or C) in the nucleic acid sequence of the TET2 gene.
In yet another embodiment, when the PAM sequence recognized by the editing protein is 5'-NNGRR-3', 5'-NNGRRT-3' or/and 5'-NNGRRV-3' (R ═ a or G; V ═ G, C or a; N ═ A, T, G or C, or A, U, G or C), the target sequence may be a contiguous 10bp-25bp nucleotide sequence of the 5 'end or/and 3' end of 5'-NNGRR-3', 5'-NNGRRT-3' or/and 5'-NNGRRV-3' (R ═ a or G; V ═ G, C or a; N ═ A, T, G or C, or A, U, G or C) in the nucleic acid sequence adjacent to the TET2 gene.
In embodiments, when the PAM sequence recognized by the editing protein is 5'-TTN-3' (N ═ A, T, G or C; or A, U, G or C), the target sequence may be a contiguous 10bp-25bp nucleotide sequence adjacent to the 5 'end or/and the 3' end of 5'-TTN-3' (N ═ A, T, G or C; or A, U, G or C) in the nucleic acid sequence of the TET2 gene.
In another example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence adjacent to the 5 'end or/and 3' end of the PAM sequence in the nucleic acid sequence of the PSGL-1 gene.
In embodiments, when the PAM sequence recognized by the editing protein is 5'-NGG-3', 5'-NAG-3' or/and 5'-NGA-3' (N ═ A, T, G or C; or A, U, G or C), the target sequence may be a contiguous 10bp-25bp nucleotide sequence at the 5 'end or/and the 3' end of 5'-NGG-3', 5'-NAG-3' or/and 5'-NGA-3' (N ═ A, T, G or C; or A, U, G or C) in the nucleic acid sequence adjacent to the PSGL-1 gene.
In another embodiment, when the PAM sequence recognized by the editing protein is 5'-NGGNG-3', 5 '-NNAGAAW-W-3' (W ═ a or T; N ═ A, T, G or C, or A, U, G or C), the target sequence may be a contiguous 10bp-25bp nucleotide sequence at the 5 'end or/and the 3' end of 5'-NGGNG-3', 5 '-NNAGAAW-W-3' (W ═ a or T; N ═ A, T, G or C, or A, U, G or C) in the nucleic acid sequence adjacent to the PSGL-1 gene.
In yet another embodiment, when the PAM sequence recognized by the editing protein is 5' -NNNNGATT-3' or/and 5' -NNNGCTT-3' (N ═ A, T, G or C; or A, U, G or C), the target sequence may be a contiguous 10bp-25bp nucleotide sequence of the 5' -nngatt-3 ' or/and 5' -NNNGCTT-3' (N ═ A, T, G or C; or A, U, G or C) end or/and 3' end in the nucleic acid sequence adjacent to the PSGL-1 gene.
In embodiments, when the PAM sequence recognized by the editing protein is 5'-NNNVRYAC-3' (V ═ G, C or a; R ═ a or G; Y ═ C or T; N ═ A, T, G or C, or A, U, G or C), the target sequence may be a contiguous 10bp-25bp nucleotide sequence of the 5'-NNNVRYAC-3' (V ═ G, C or a; R ═ a or G; Y ═ C or T; N ═ A, T, G or C, or A, U, G or C) at the 5 'end or/and the 3' end of the nucleic acid sequence adjacent to the PSGL-1 gene.
In another embodiment, when the PAM sequence recognized by the editing protein is 5'-NAAR-3' (R ═ a or G; N ═ A, T, G or C, or A, U, G or C), the target sequence may be a contiguous 10bp-25bp nucleotide sequence adjacent to the 5 'end or/and the 3' end of 5'-NAAR-3' (R ═ a or G; N ═ A, T, G or C, or A, U, G or C) in the nucleic acid sequence of the PSGL-1 gene.
In yet another embodiment, when the PAM sequence recognized by the editing protein is 5'-NNGRR-3', 5'-NNGRRT-3' or/and 5'-NNGRRV-3' (R ═ a or G; V ═ G, C or a; N ═ A, T, G or C, or A, U, G or C), the target sequence may be a contiguous 10bp-25bp nucleotide sequence of the 5 'end or/and 3' end of 5'-NNGRR-3', 5'-NNGRRT-3' or/and 5'-NNGRRV-3' (R ═ a or G; V ═ G, C or a; N ═ A, T, G or C, or A, U, G or C) in the nucleic acid sequence adjacent to the PSGL-1 gene.
In embodiments, when the PAM sequence recognized by the editing protein is 5'-TTN-3' (N ═ A, T, G or C; or A, U, G or C), the target sequence may be a contiguous 10bp-25bp nucleotide sequence adjacent to the 5 'end or/and the 3' end of 5'-TTN-3' (N ═ A, T, G or C; or A, U, G or C) in the nucleic acid sequence of the PSGL-1 gene.
In yet another example, the target sequence may be a contiguous 10bp-25bp nucleotide sequence adjacent to the 5 'end or/and 3' end of a motif (PAM) of a pre-spacer sequence in a nucleic acid sequence adjacent to the KDM6A gene.
In embodiments, when the PAM sequence recognized by the editing protein is 5'-NGG-3', 5'-NAG-3' or/and 5'-NGA-3' (N ═ A, T, G or C; or A, U, G or C), the target sequence may be a contiguous 10bp-25bp nucleotide sequence of the 5 'end or/and 3' end of 5'-NGG-3', 5'-NAG-3' or/and 5'-NGA-3' (N ═ A, T, G or C; or A, U, G or C) in the nucleic acid sequence adjacent to KDM6A gene.
In another embodiment, when the PAM sequence recognized by the editing protein is 5'-NGGNG-3' or/and 5 '-NNAGAAW-W-3' (W ═ a or T; N ═ A, T, G or C, or A, U, G or C), the target sequence may be a contiguous 10bp-25bp nucleotide sequence at the 5 'end or/and 3' end of 5'-NGGNG-3' or/and 5 '-NNAGAAW-W-3' (W ═ a or T; N ═ A, T, G or C, or A, U, G or C) in the nucleic acid sequence adjacent to KDM6A gene.
In yet another embodiment, when the PAM sequence recognized by the editing protein is 5' -NNNNGATT-3' or/and 5' -NNNGCTT-3' (N ═ A, T, G or C; or A, U, G or C), the target sequence may be a contiguous 10bp-25bp nucleotide sequence of the 5' -nngatt-3 ' or/and 5' -NNNGCTT-3' (N ═ A, T, G or C; or A, U, G or C) end or/and 3' end in the nucleic acid sequence adjacent to KDM6A gene.
In embodiments, when the PAM sequence recognized by the editing protein is 5' -NNNVRYAC-3' (V ═ G, C or a; R ═ a or G; Y ═ C or T; N ═ A, T, G or C, or A, U, G or C), the target sequence may be a contiguous 10bp-25bp nucleotide sequence of the 5' -NNNVRYAC-3' (V ═ G, C or a; R ═ a or G; Y ═ C or T; N ═ A, T, G or C, or A, U, G or C) or/and the 3' end of the nucleic acid sequence adjacent to KDM6A gene.
In another embodiment, when the PAM sequence recognized by the editing protein is 5'-NAAR-3' (R ═ a or G; N ═ A, T, G or C, or A, U, G or C), the target sequence may be a contiguous 10bp-25bp nucleotide sequence adjacent to the 5 'end or/and the 3' end of 5'-NAAR-3' (R ═ a or G; N ═ A, T, G or C, or A, U, G or C) in the nucleic acid sequence of KDM6A gene.
In yet another embodiment, when the PAM sequence recognized by the editing protein is 5' -NNGRR-3', 5' -NNGRRT-3' or/and 5' -NNGRRV-3' (R ═ a or G; V ═ G, C or a; N ═ A, T, G or C, or A, U, G or C), the target sequence may be a contiguous 10bp-25bp nucleotide sequence of the 5' -grnnr-3 ', 5' -NNGRRT-3' or/and 5' -NNGRRV-3' (R ═ a or G; V ═ G, C or a; N ═ A, T, G or C, or A, U, G or C) end or/and the 3' end of the nucleic acid sequence adjacent to KDM6A gene.
In embodiments, when the PAM sequence recognized by the editing protein is 5'-TTN-3' (N ═ A, T, G or C; or A, U, G or C), the target sequence may be a contiguous 10bp-25bp nucleotide sequence adjacent to the 5 'end or/and the 3' end of 5'-TTN-3' (N ═ A, T, G or C; or A, U, G or C) in the nucleic acid sequence of KDM6A gene.
Examples of target sequences that may be used in one embodiment of the invention are tabulated below, the target sequences described in the table being guide nucleic acid non-binding sequences; complementary sequences, i.e., the leader nucleic acid binding sequence can be predicted from the sequence.
[ Table 1] target sequences of immunoregulatory genes
Figure BDA0002359848460000691
Figure BDA0002359848460000701
Figure BDA0002359848460000711
Figure BDA0002359848460000721
Figure BDA0002359848460000731
Figure BDA0002359848460000741
Figure BDA0002359848460000751
Figure BDA0002359848460000761
Figure BDA0002359848460000771
Figure BDA0002359848460000781
Aspects of the present disclosure relate to gene manipulation compositions for the manual manipulation of immunomodulatory genes.
Compositions for gene manipulation can be used to produce artificially modified immunomodulatory genes. In addition, immunomodulatory genes artificially modified by gene-manipulated compositions can modulate the immune system.
The term "artificially modified or engineered or artificially engineered" refers to a state in which an artificial modification is applied, not an original state existing in a natural state. The following artificially modified or engineered non-natural immunomodulatory genes may be used interchangeably with artificial immunomodulatory genes.
The "immune system" of the present invention is a term encompassing all phenomena affecting the immune response in vivo (i.e., participating in the mechanism that exhibits new immune potency) through the altered function of manipulated immune modulatory factors, including all materials, compositions, methods and uses that are directly or indirectly involved in such immune systems. For example, the immune system includes all genes, immune cells and immune organs/tissues involved in innate, adaptive, cellular, humoral, active and passive immune responses.
The compositions for gene manipulation disclosed herein can comprise a guide nucleic acid and an editing protein.
Compositions for gene manipulation may comprise:
(a) a guide nucleic acid that can form a complementary binding with a target sequence of an immune modulatory gene or a nucleic acid sequence encoding the same; and
(b) one or more editing proteins or nucleic acid sequences encoding the same.
The explanation about the above-mentioned immunoregulatory gene is as described above.
The explanation for the above target sequence is as described above.
Compositions for gene manipulation may comprise a guide nucleic acid-editing protein complex.
The term "guide nucleic acid-editing protein complex" refers to a complex formed by the interaction between a guide nucleic acid and an editing protein.
The explanation about the above guide nucleic acid is as described above.
An "editing protein" refers to a peptide, polypeptide, or protein that is capable of interacting with a nucleic acid, either directly or without direct binding.
In this case, the nucleic acid may be a target nucleic acid, a gene, or a nucleic acid contained in a chromosome. Here, the nucleic acid may be a guide nucleic acid.
The editing protein may be an enzyme.
Herein, the term "enzyme" refers to a polypeptide or protein containing a domain capable of cleaving a nucleic acid, gene, or chromosome.
The enzyme may be a nuclease or a restriction enzyme.
Editing proteins may include enzymes with full activity.
Herein, "enzyme having full activity" refers to an enzyme having the same function as the original function of a wild-type enzyme that cleaves a nucleic acid, gene or chromosome. For example, a wild-type enzyme that cleaves double-stranded DNA can be a fully active enzyme that cleaves all double-stranded DNA. In another example, when a partial amino acid sequence of a wild-type enzyme that cleaves double-stranded DNA is deleted or substituted by artificial modification, if the artificially modified enzyme mutant cleaves double-stranded DNA identically to the wild-type enzyme, the artificially modified enzyme mutant may be an enzyme having full activity.
Further, the enzyme having full activity may include an enzyme having an improved function as compared to that of a wild-type enzyme. For example, a particular modified or engineered form of a wild-type enzyme that cleaves double-stranded DNA may have improved full enzyme activity compared to the wild-type enzyme, i.e., improved activity for cleaving double-stranded DNA.
Editing proteins may include enzymes with incomplete or partial activity.
Herein, the term "enzyme having incomplete or partial activity" refers to an enzyme having only a partial original function of a wild-type enzyme that cleaves a nucleic acid, a gene, or a chromosome. For example, a particular modified or engineered form of the wild-type enzyme that cleaves double-stranded DNA may be a form having a first function or a form having a second function. Here, the first function may be a function of cleaving a first strand of the double-stranded DNA, and the second function may be a function of cleaving a second strand of the double-stranded DNA. Here, the enzyme having the first function or the enzyme having the second function may be an enzyme having incomplete or partial activity.
The editing protein may comprise an inactivated enzyme.
Herein, the term "inactivated enzyme" refers to an enzyme in which the original function of a wild-type enzyme that cleaves a nucleic acid, gene, or chromosome is completely inactivated. For example, a particular modified or engineered form of the wild-type enzyme may be one that loses both the first and second functions, i.e., both the first function to cleave the first strand of double-stranded DNA and the second function to cleave the second strand of double-stranded DNA. Here, the enzyme that loses the first function and the second function may be an inactivated enzyme.
The editing protein may be a fusion protein.
Herein, a fusion protein refers to a protein produced by fusing an enzyme to an additional domain, peptide, polypeptide, or protein.
The additional domain, peptide, polypeptide or protein may be a functional domain, peptide, polypeptide or protein having the same or different function as the functional domain, peptide, polypeptide or protein comprised in the enzyme.
The fusion protein may comprise additional functional domains, peptides, polypeptides, or proteins at one or more regions at or near the amino terminus of the enzyme, at or near the carboxy terminus of the enzyme, in an intermediate portion of the enzyme, and combinations thereof.
Here, the functional domain, peptide, polypeptide or protein may be a domain, peptide, polypeptide or protein having methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity or nucleic acid binding activity, or a tag or reporter gene for purifying and isolating proteins (including peptides), but the present invention is not limited thereto.
The functional domain, peptide, polypeptide or protein may be a deaminase.
The tags include histidine (His) tag, V5 tag, FLAG tag, influenza Hemagglutinin (HA) tag, Myc tag, VSV-G tag, and thioredoxin (Trx) tag, and the reporter genes include Glutathione Sulfurtransferase (GST), horseradish peroxidase (HRP), Chloramphenicol Acetyltransferase (CAT), β -galactosidase, β -glucuronidase, luciferase, autofluorescent proteins including Green Fluorescent Protein (GFP), HcRed, DsRed, Cyan Fluorescent Protein (CFP), Yellow Fluorescent Protein (YFP), and Blue Fluorescent Protein (BFP), but the present invention is not limited thereto.
Furthermore, the functional domain, peptide, polypeptide or protein may be a nuclear localization sequence or signal (NLS) or a nuclear export sequence or signal (NES).
The NLS can be NLS of SV40 virus large T antigen with amino acid sequence PKKKRKV, NLS derived from nucleoplasmin (such as bimolecular Nucleoplasmin (NLS) with sequence KRPAATKKAGQAKKKK), c-myc NLS with amino acid sequence PAAKRVKLD or RQRRNELKRSP, hRNPA 1M 9 NLS with sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY, IBB domain sequence RMRIZFKGKNTAELRRVTERVTVELKRVEKLKDEQKRRNV derived from import protein α (immunoprotein- α), myoma T protein sequence VSRKRPRP and PPKKARED, human p53 sequence POPKKKPL, mouse c-abl IV sequence SALIKKKKKMAP, influenza virus NS1 sequence DRLRR and PKQKKRK, hepatitis virus delta antigen sequence RKLKKKIKKL, mouse Mx1 protein sequence REKKKFLKRR, human multimeric (ADPyKKRK) polymerase sequence KRKGDEVDGVDEVAKKKSKK, human glucocorticoid receptor (ADP) sequence RKCLQAGMNLEARKTKK, but the invention is not limited to human glucocorticoid receptor sequence RKCLQAGMNLEARKTKK.
The additional domain, peptide, polypeptide, or protein may be a dysfunctional domain, peptide, polypeptide, or protein that does not perform a particular function. Here, the dysfunctional domain, peptide, polypeptide or protein may be a domain, peptide, polypeptide or protein that does not affect the function of the enzyme.
The fusion protein may comprise additional dysfunctional domains, peptides, polypeptides, or proteins at or near the amino terminus of the enzyme, at or near the carboxy terminus of the enzyme, in the middle of the enzyme, and combinations thereof.
The editing protein may be a native enzyme or a fusion protein.
The editing protein may be present as a partially modified native enzyme or as a fusion protein.
The editing protein may be an artificially produced enzyme or fusion protein that does not exist in the natural state.
The editing protein may exist in the form of a partially modified artificial enzyme or a fusion protein that does not exist in the natural state.
Here, the modification may be substitution, deletion, addition, or a combination of the above modifications of amino acids contained in the editing protein.
In addition, the modification may be substitution, deletion, addition, or a combination of the above modifications of a part of bases in the base sequence encoding the editing protein.
In addition, the composition for gene manipulation may optionally further comprise a donor comprising a specific nucleotide sequence desired to be inserted or a nucleic acid sequence encoding the donor.
Here, the nucleotide sequence desired to be inserted may be a partial nucleotide sequence in a gene involved in immunity.
Here, the nucleotide sequence desired to be inserted may be a nucleotide sequence modified or introduced by mutation of an immune regulatory gene subjected to manipulation.
The term "donor" refers to a nucleic acid sequence of a gene or nucleic acid that helps repair damage by HDR.
The donor may be a double-stranded nucleic acid or a single-stranded nucleic acid.
The donor may be linear or circular.
The donor may comprise a nucleic acid sequence having homology to the target gene or nucleic acid.
For example, the donor may comprise nucleotide sequences having homology with the nucleotide sequences at the positions to be inserted into the specific nucleic acid (e.g., upstream and downstream of the damaged nucleic acid), respectively. Specifically, the specific nucleic acid to be inserted may be located between a nucleotide sequence having homology with the downstream nucleotide sequence of the damaged nucleic acid and a nucleotide sequence having homology with the upstream nucleotide sequence of the damaged nucleic acid. Specifically, the nucleotide sequences having the above homology may have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% or more homology or complete homology.
The donor may optionally comprise additional nucleotide sequences. In particular, the additional nucleotide sequence may play a role in enhancing the HDR efficiency, stability, or knock-in efficiency of the donor.
For example, the additional nucleotide sequence may be an a and T base-rich nucleotide sequence (i.e., an a-T-rich domain). Alternatively, the additional nucleotide sequence may be a scaffold/matrix attachment region (S/MAR).
The guide nucleic acids, editing proteins, or guide nucleic acid-editing protein complexes disclosed herein can be delivered or introduced into a subject in a variety of forms.
The term "subject" refers to an organism into which a guide nucleic acid, editing protein, or guide nucleic acid-editing protein complex is introduced; organisms in which (operates) guide nucleic acids, editing proteins or guide nucleic acid-editing protein complexes are operated; or a specimen or sample obtained from said organism.
The subject may be an organism comprising a target gene or chromosome that directs the nucleic acid-editing protein complex.
The organism can be an animal, an animal tissue, or an animal cell.
The organism may be a human, human tissue or human cells.
The tissue may be eye, skin, liver, kidney, heart, lung, brain, muscle or blood.
The cell can be an immune cell, such as a natural killer cell (NK cell), a T cell, a B cell, a dendritic cell, and a macrophage or stem cell.
A sample or specimen can be obtained from an organism (e.g., saliva, blood, liver tissue, brain tissue, hepatocytes, neurons, phagocytic cells, T cells, B cells, astrocytes, cancer cells, or stem cells) that contains a target gene or chromosome.
Preferably, the subject may be an organism comprising an immunomodulatory gene.
The guide nucleic acid, editing protein, or guide nucleic acid-editing protein complex may be delivered into or introduced into the subject in DNA, RNA, or mixed form.
Here, DNA, RNA or mixtures thereof encoding the guide nucleic acid and/or editing protein may be delivered or introduced into the subject by methods known in the art.
Alternatively, the DNA, RNA, or mixtures thereof encoding the guide nucleic acid and/or editing protein may be delivered or introduced into the subject via a vector, a non-vector, or a combination thereof.
The vector may be a viral vector or a non-viral vector (e.g., a plasmid).
The non-carrier can be naked DNA, a DNA complex, or mRNA.
Nucleic acid sequences encoding the guide nucleic acid and/or editing protein can be delivered into or introduced into a subject by means of a vector.
The vector may comprise a nucleic acid sequence encoding a guide nucleic acid and/or an editing protein.
For example, the vector may comprise both nucleic acid sequences encoding the guide nucleic acid and the editing protein, respectively.
For example, the vector may comprise a nucleic acid sequence encoding a guide nucleic acid.
As an example, the domains comprised in the guide nucleic acid may all be comprised in one vector, or may be divided and subsequently comprised in different vectors.
For example, a vector may comprise a nucleic acid sequence encoding an editing protein.
In one example, in the case of an editing protein, the nucleic acid sequence encoding the editing protein may be contained in one vector, or it may be divided and then contained in several vectors.
The carrier may contain one or more regulatory/control components.
Here, the regulating/controlling component may include: promoters, enhancers, introns, polyadenylation signals, Kozak consensus sequences, Internal Ribosome Entry Sites (IRES), splice acceptors, and/or 2A sequences.
The promoter may be a promoter recognized by RNA polymerase II.
The promoter may be a promoter recognized by RNA polymerase III.
The promoter may be an inducible promoter.
The promoter may be a subject-specific promoter.
The promoter may be a viral or non-viral promoter.
With respect to the promoter, an appropriate promoter may be used depending on the control region (i.e., the nucleic acid sequence encoding the guide nucleic acid or the editing protein).
For example, a promoter that can be used to direct a nucleic acid can be the H1, EF-1a, tRNA, or U6 promoter. For example, the promoter that can be used to edit the protein can be the CMV, EF-1a, EFS, MSCV, PGK, or CAG promoter.
The vector may be a viral vector or a recombinant viral vector.
The virus may be a DNA virus or an RNA virus.
Here, the DNA virus may be a double stranded DNA (dsdna) virus or a single stranded DNA (ssdna) virus.
Here, the RNA virus may be a single-stranded RNA (ssrna) virus.
The virus may be a retrovirus, lentivirus, adenovirus, adeno-associated virus (AAV), vaccinia virus, poxvirus, or herpes simplex virus, but the present invention is not limited thereto.
In general, a virus can infect a host (e.g., a cell), thereby introducing nucleic acid encoding genetic information of the virus into the host or inserting nucleic acid encoding genetic information into the host genome. Viruses with such characteristics can be used to introduce guide nucleic acids and/or editing proteins into a subject. The introduced guide nucleic acid and/or editing protein using the virus can be transiently expressed in a subject (e.g., a cell). Alternatively, the use of a virally introduced guide nucleic acid and/or editing protein may be expressed in a subject (e.g., a cell) for a prolonged period of time (e.g., 1, 2, or 3 weeks, 1, 2, 3, 6, or 9 months, 1 or 2 years, or permanently).
Depending on the type of virus, the packaging capacity of the virus may vary from at least 2kb to 50 kb. Depending on such packaging capabilities, a viral vector comprising a guide nucleic acid or an editing protein or a viral vector comprising both a guide nucleic acid and an editing protein may be designed. Alternatively, viral vectors can be designed that contain guide nucleic acids, editing proteins, and additional components.
In one example, a recombinant lentivirus can be used to deliver or introduce nucleic acid sequences encoding a guide nucleic acid and/or editing a protein.
In another example, a nucleic acid sequence encoding a guide nucleic acid and/or editing protein can be delivered or introduced using a recombinant adenovirus.
In yet another embodiment, a nucleic acid sequence encoding a guide nucleic acid and/or editing protein can be delivered or introduced using a recombinant AAV.
In yet another example, a nucleic acid sequence encoding a guide nucleic acid and/or editing a protein can be delivered or introduced using a mixed virus (e.g., a mixture of one or more of the viruses listed herein).
Non-vectors can be used to deliver or introduce nucleic acid sequences encoding guide nucleic acids and/or editing proteins into a subject.
The non-vector may comprise a nucleic acid sequence encoding a guide nucleic acid and/or an editing protein.
The non-carrier can be naked DNA, a DNA complex, mRNA, or a mixture thereof.
Non-carriers can be delivered into or introduced into a subject by electroporation, particle bombardment, sonoporation, magnetic transfection, transient cell compression or compaction (e.g., as described in Lee et al (2012) NanoLett., 12, 6322-.
As an example, delivery via electroporation may be performed by mixing cells with nucleic acid sequences encoding guide nucleic acids and/or editing proteins in a cartridge (cartridge), chamber (chamber), or cuvette (cuvette) and applying electrical stimulation to the cells for a predetermined duration and amplitude.
In another example, the non-carrier can be delivered using nanoparticles. The nanoparticles may be inorganic nanoparticles (e.g., magnetic nanoparticles, silica, etc.) or organic nanoparticles (e.g., polyethylene glycol (PEG) -coated lipids, etc.). The outer surface of the nanoparticle may be conjugated with a positively charged polymer (e.g., polyethyleneimine, polylysine, polyserine, etc.) capable of attachment.
In certain embodiments, the non-carrier can be delivered using a lipid shell.
In certain embodiments, non-vectors may be delivered using exosomes (exosomes). Exosomes are endogenous nanovesicles that transfer proteins and RNA and can deliver RNA to the brain and another target organ.
In certain embodiments, the non-carrier can be delivered using liposomes. Liposomes are spherical vesicular structures consisting of a single or multiple lamellar lipid bilayers surrounding an inner aqueous chamber, and a relatively impermeable outer lipophilic phospholipid bilayer. Although liposomes can be made from several different types of lipids, phospholipids are most commonly used to produce liposomes as drug carriers.
In addition, the compositions for non-carrier delivery may contain other additives.
The editing protein may be delivered or introduced into the subject in the form of a peptide, polypeptide or protein.
The editing protein may be delivered or introduced into a subject in the form of a peptide, polypeptide or protein by methods known in the art.
The peptide, polypeptide, or protein form can be delivered into or introduced into the subject by electroporation, microinjection, transient cell compression or extrusion (e.g., as described in the document "Lee et al (2012) nanolett, 12, 6322-.
The peptide, polypeptide or protein may be delivered with a nucleic acid sequence encoding a guide nucleic acid.
In one example, delivery via electroporation can be performed by mixing cells into which the editing proteins are to be introduced with (or without) a guide nucleic acid in a cartridge, chamber, or cuvette and applying an electrical stimulus to the cells for a predetermined duration and amplitude.
The guide nucleic acid and editing protein can be delivered into or introduced into the subject in the form of a nucleic acid-protein mixture.
The guide nucleic acid and editing protein may be delivered into or introduced into the subject in the form of a guide nucleic acid-editing protein complex.
For example, the guide nucleic acid may be DNA, RNA, or a mixture thereof. The editing protein may be a peptide, polypeptide or protein.
In one example, the guide nucleic acid and the editing protein may be delivered into or introduced into the subject in the form of a guide nucleic acid-editing protein complex comprising an RNA-based guide nucleic acid and a protein-based editing protein, i.e., a Ribonucleoprotein (RNP).
The guide nucleic acid-editing protein complexes disclosed herein can modify a target nucleic acid, gene, or chromosome.
For example, the guide nucleic acid-editing protein complex induces a modification to the sequence of a target nucleic acid, gene, or chromosome. Thus, proteins expressed by a target nucleic acid, gene, or chromosome may have their modified structure and/or function, their controlled expression, or their deleted expression.
The guide nucleic acid-editing protein complex may function at the DNA, RNA, gene, or chromosome level.
In one example, the guide nucleic acid-editing protein complex can be used to modulate (e.g., inhibit, repress, reduce, increase, or facilitate) expression of a protein encoded by a target gene, or to modulate (e.g., inhibit, repress, reduce, increase, or facilitate) activity of a protein, or to express a modified protein by engineering or modifying the target gene.
The guide nucleic acid-editing protein complex may play a role in the gene transcription and translation stages.
In one example, the guide nucleic acid-editing protein complex can promote or repress transcription of a target gene, thereby modulating (e.g., inhibiting, repressing, reducing, increasing, or promoting) expression of a protein encoded by the target gene.
In another example, the guide nucleic acid-editing protein complex can promote or repress translation of the target gene, thereby modulating (e.g., inhibiting, repressing, reducing, increasing, or promoting) expression of the protein encoded by the target gene.
In embodiments disclosed herein, a composition for gene manipulation can comprise a gRNA and a CRISPR enzyme.
Compositions for gene manipulation may comprise:
(a) a gRNA capable of forming a complementary binding to a target sequence of an immunomodulatory gene or a nucleic acid sequence encoding the target sequence of the immunomodulatory gene; and
(b) one or more CRISPR enzymes or nucleic acid sequences encoding the CRISPR enzymes.
The explanation about the above-mentioned immunoregulatory gene is as described above.
The explanation for the above target sequence is as described above.
A genetically manipulated composition can include a gRNA-CRISPR enzyme complex.
The term "gRNA-CRISPR enzyme complex" refers to a complex formed by the interaction between a gRNA and a CRISPR enzyme.
Explanations for the above grnas are as described above.
A "CRISPR enzyme" is the main protein component of the CRISPR-Cas system, which forms the CRISPR-Cas system by forming a complex with the gRNA.
A CRISPR enzyme can be a nucleic acid or polypeptide (or protein) having a sequence encoding a CRISPR enzyme.
The CRISPR enzyme may be a type II CRISPR enzyme.
The crystal structure of type II CRISPR enzymes was determined from studies on two or more types of native microorganism type II CRISPR enzyme molecules (Jinek et al, Science, 343 (6176): 1247997, 2014) and on the complexation of Streptococcus pyogenes Cas9(SpCas9) with gRNAs (Nishimasu et al, Cell, 156: 935-949, 2014; and Anders et al, Nature, 2014, doi: 10.1038/Nature 13579).
Type II CRISPR enzymes comprise two leaves (lobes), a Recognition (REC) leaf and a Nuclease (NUC) leaf, each leaf comprising several domains.
REC leaves contain an arginine-rich helical Bridge (BH) domain, a REC1 domain, and a REC2 domain.
Here, the BH domain is a long α helix and is an arginine-rich region, while the REC1 domain and REC2 domain play important roles in recognizing double strands formed in grnas (e.g., single-stranded grnas, double-stranded grnas, or tracrrnas).
NUC leaves contain a RuvC domain, a HNH domain, and a PAM Interaction (PI) domain. Here, RuvC domain includes RuvC-like domain, or HNH domain is used to include HNH-like domain.
Here, the RuvC domain shares structural similarity with members of the naturally occurring microbial family with type II CRISPR enzymes and cleaves single strands (e.g., non-complementary strands of a target gene or nucleic acid, i.e., strands that do not form complementary binding with the gRNA). In the art, RuvC domain sometimes refers to RuvCI domain, RuvCII domain, or RuvCIII domain, commonly referred to as RuvCI, RuvCII, or RuvCIII.
The HNH domain shares structural similarity with HNH endonucleases and cleaves a single strand (e.g., the complementary strand of the target nucleic acid molecule, i.e., the strand that forms a complementary bond with the gRNA). The HNH domain is located between RuvCII and III motifs.
The PI domain recognizes or interacts with a specific nucleotide sequence (i.e., a promiscuous sequence adjacent motif (PAM)) in a target gene or nucleic acid. Here, PAM can vary depending on the source of the type II CRISPR enzyme. For example, when the CRISPR enzyme is SpCas9, the PAM can be 5 '-NGG-3'; when the CRISPR enzyme is streptococcus thermophilus Cas9(StCas9), the PAM can be 5'-NNAGAAW-3' (W ═ a or T); when the CRISPR enzyme is neisseria meningitidis Cas9(NmCas9), the PAM can be 5 '-NNNNGATT-3'; when the CRISPR enzyme is campylobacter jejuni Cas9(CjCas9), the PAM can be 5'-NNNVRYAC-3' (V ═ G or C or a; R ═ a or G; Y ═ C or T), where N can be A, T, G or C, or A, U, G or C. However, while it is generally understood that PAM is determined according to the source of the enzyme as described above, PAM can vary as research progresses for mutants of the enzyme of that source.
The type II CRISPR enzyme may be Cas 9.
Cas9 can be derived from various microorganisms: for example, Streptococcus pyogenes (Streptococcus pyogenenes), Streptococcus thermophilus (Streptococcus thermophilus), Streptococcus sp (Streptococcus sp.), Staphylococcus aureus (Staphylococcus aureus), Streptomyces dawdansi (Nocardia dasson), Streptomyces pristinaespiralis, Streptomyces viridochromogenes (Streptomyces griseus), Streptomyces roseosporum (Streptomyces roseosporum), Bacillus acidocaldarius (Alicyclobacillus acidocaldarius), Bacillus pseudolyticus (Bacillus pseudomonads), Bacillus subtilis, Bacillus acidolyticus, Lactobacillus salivarius, Bacillus subtilis, Clostridium (Clostridium sp), Bacillus mucilaginosus (Bacillus sp), Bacillus mucilaginosus (Bacillus mucilaginosus), Bacillus mucilaginosus strain, Bacillus mucilaginosus, Bacillus subtilis, Bacillus mucilaginosus, Bacillus subtilis, micromonas diminuta (Finegoldia magna), Natranobiusthermophilus, Pelotomaculum thermoproprionicus, Acidithiobacillus caldus (Acidithiobacillus caldus), Acidithiobacillus ferrooxidans (Acidithiobacillus ferrooxidans), Allochinousness, Hippocampus (Marinobacter sp.), Nitrosococcus halophthalicus, Nitrosococcus watsonii, Pseudocalophyllobacter haloplanktis, Kteobabacter ramis, Methanolobium vestigiatum, Anabaena varia (Anabaena variabilis), Synechococcus foameus (Nodularia), Nodularia sp, Thermoascus sp, Arthrospira sp, Anabaena sp, Thermoascus sp, and Nodularia sp.
Cas9, an enzyme that binds to a gRNA in order to cleave or modify a target sequence or location on a target gene or nucleic acid, can consist of an HNH domain (capable of cleaving a nucleic acid strand that forms a complementary binding with the gRNA), a RuvC domain (capable of cleaving a nucleic acid strand that forms a non-complementary binding with the gRNA), a REC domain (recognizing a target), and a PI domain (recognizing a PAM). For specific structural features of Cas9, see Hiroshi Nishimasu et al, (2014) Cell 156: 935-949.
Cas9 may be isolated from naturally occurring microorganisms or produced non-naturally by recombinant or synthetic methods.
Further, the CRISPR enzyme can be a type V CRISPR enzyme.
Type V CRISPR enzymes contain a similar RuvC domain (corresponding to that of type II CRISPR enzymes) and may consist of a Nuc domain (rather than the HNH domain of type II CRISPR enzymes), REC and WED domains (interacting with the target) and a PI domain (recognizing PAM). For specific structural features of type V CRISPR enzymes, see TakashiYamano et al (2016) Cell 165: 949-962.
The type V CRISPR enzyme can interact with the gRNA to form a gRNA-CRISPR enzyme complex, i.e., a CRISPR complex, and can allow the guide sequence to access a target sequence comprising a PAM sequence under the cooperation of the gRNA. Here, the ability of the type V CRISPR enzyme to interact with a target gene or nucleic acid depends on the PAM sequence.
The PAM sequence is a sequence present in a target gene or nucleic acid that is recognized by the PI domain of a V-type CRISPR enzyme. The PAM sequence may vary depending on the source of the type V CRISPR enzyme. That is, depending on the species, there are different PAM sequences that can be specifically recognized. For example, the PAM sequence identified by Cpf1 may be 5'-TTN-3' (N is A, T, C or G). However, while it is generally understood that PAM is determined according to the source of the enzyme as described above, PAM can vary as research progresses for mutants of the enzyme of that source.
The type V CRISPR enzyme may be Cpf 1.
Cpf1 may be Cpf1 derived from: streptococcus (Streptococcus), Campylobacter (Campylobacter), Nitratifractor, Staphylococcus (Staphylococcus), Parvibacterium, Roseburia (Roseburia), Neisseria (Neisseria), Acetobacter gluconicum (Gluconobacter), Azospirillum (Azospirillum), Sphaechaeta, Lactobacillus (Lactobacillus), Eubacterium (Eubacterium), Corynebacterium (Corynebacterium), Carnobacterium (Carnobacterium), Rhodobacterium (Rhodobacterium), Listeria (Listeria), Paluobacter, Clostridium (Clostridium), Lactobacilli (Lachnospiricus), clostridium, Cellulosidium (Leptotrichia), Francisella (Francisella), Legionella (Legionella), Alicyclobacillus (Alicyclobacillus), Methanomethyophilus, Porphyromonas (Porphyromonas), Prevotella (Prevotella), Bacteroides (Bacteroides), Sporococcus (Helcococcus), Letospira (Letospira), Desulovibrio (Desulfovibrio), Desufoninum, Torulopsis (Opitutaceae), Bacillus tumefaciens (Tuberibacillus), Bacillus (Bacillus), Bacillus brevis (Brevibacillus), Methylobacterium (Methylobacterium), or amino acid (Acidaminococcus).
Cpf1 comprises a RuvC domain (similar to and corresponding to the RuvC domain of type II CRISPR enzymes) and may consist of a Nuc domain (rather than the HNH domain of Cas9), REC and WED domains (interacting with the target) and a PI domain (recognizing PAM). For specific structural features of Cpf1, see Takashi Yamano et al (2016) Cell 165: 949-962.
Cpf1 may be isolated from naturally occurring microorganisms or non-naturally produced by recombinant or synthetic methods.
CRISPR enzymes can be nucleases or restriction enzymes that have the function of cleaving the double strand of the target gene or nucleic acid.
The CRISPR enzyme can be a CRISPR enzyme with full activity.
Here, "having full activity" refers to a state having the same function as a wild-type CRISPR enzyme, and the CRISPR enzyme in this state is referred to as "a CRISPR enzyme having full activity". Here, the "function of the wild-type CRISPR enzyme" refers to a state having a function of cleaving a double-stranded DNA, that is, a state having a first function of cleaving a first strand of the double-stranded DNA and a second function of cleaving a second strand of the double-stranded DNA.
A CRISPR enzyme with full activity can be a wild-type CRISPR enzyme that cleaves double-stranded DNA.
A CRISPR enzyme with full activity can be a CRISPR enzyme mutant in which the wild-type CRISPR enzyme that cleaves double-stranded DNA is modified or manipulated.
A CRISPR enzyme mutant can be an enzyme that has one or more amino acids substituted for another amino acid or one or more amino acids deleted from the amino acid sequence of a wild-type CRISPR enzyme.
The CRISPR enzyme mutant may be an enzyme in which one or more amino acids are added to the amino acid sequence of a wild-type CRISPR enzyme. Here, the position of the added amino acid may be N-terminal, C-terminal, or within the amino acid sequence of the wild-type enzyme.
The CRISPR enzyme mutants can be fully active enzymes with improved function compared to wild-type CRISPR enzymes.
For example, a particular modified or manipulated form of a wild-type CRISPR enzyme (i.e., a CRISPR enzyme mutant) can cleave double-stranded DNA without binding to the double-stranded DNA to be cleaved or remaining at a distance. In this case, the modified or manipulated form may be a fully active CRISPR enzyme with improved function compared to the wild type CRISPR enzyme.
The CRISPR enzyme mutant can be a fully active enzyme having reduced function as compared to a wild-type CRISPR enzyme.
For example, a particular modified or manipulated form of a wild-type CRISPR enzyme (i.e., a CRISPR enzyme mutant) can cleave double-stranded DNA at a particular distance from or closer to the double-stranded DNA to be cleaved, or in the presence of certain binding. Here, certain binding may be, for example, binding between an amino acid at a specific position of the enzyme and a DNA nucleotide sequence in a cleavage position. In this case, the modified or manipulated form may be a fully active CRISPR enzyme with reduced function compared to the wild type CRISPR enzyme.
The CRISPR enzyme may be a CRISPR enzyme with incomplete or partial activity.
The term "partially or partially active" refers to a state having a function selected from the functions of a wild-type CRISPR enzyme (i.e., a first function to cleave a first strand of a double-stranded DNA, and a second function to cleave a second strand of the double-stranded DNA). Furthermore, CRISPR enzymes with incomplete or partial activity may be referred to as nickases.
The term "nickase" refers to a CRISPR enzyme that is manipulated or modified to cleave only one strand of a target gene or nucleic acid double strand, the nickase having nuclease activity that cleaves a single strand (e.g., a strand that is not complementary to or complementary to a gRNA of the target gene or nucleic acid). Therefore, nuclease activity of two nicking enzymes is required for cleavage of double strands.
The nickase can have nuclease activity of a RuvC domain of a CRISPR enzyme. That is, the nickase may not comprise the nuclease activity of the HNH domain of the CRISPR enzyme, for which the HNH domain may be manipulated or modified.
In one example, when the CRISPR enzyme is a type II CRISPR enzyme, the nickase can be a type II CRISPR enzyme comprising a modified HNH domain.
For example, when the type II CRISPR enzyme is a wild-type SpCas9, the nickase may be a SpCas9 mutant in which residue 840 in the amino acid sequence of wild-type SpCas9 is mutated from histidine to alanine and the nuclease activity of the HNH domain is inactivated. Here, the resulting nickase (i.e., SpCas9 mutant) has the nuclease activity of the RuvC domain and is therefore capable of cleaving a non-complementary strand of the target gene or nucleic acid, i.e., a strand that does not form a complementary binding with the gRNA.
In another example, when the type II CRISPR enzyme is a wild-type CjCas9, the nickase may be a CjCas9 mutant in which residue 559 of the wild-type CjCas9 amino acid sequence is mutated from histidine to alanine and the nuclease activity of the HNH domain is inactivated. Here, the resulting nickase (i.e., CjCas9 mutant) has the nuclease activity of the RuvC domain and is therefore capable of cleaving a non-complementary strand of the target gene or nucleic acid, i.e., a strand that does not form a complementary binding with the gRNA.
In addition, the nickase can have nuclease activity of the HNH domain of the CRISPR enzyme. That is, the nickase may not comprise the nuclease activity of the RuvC domain of the CRISPR enzyme, for which the RuvC domain may be manipulated or modified.
In one example, when the CRISPR enzyme is a type II CRISPR enzyme, the nickase can be a type II CRISPR enzyme comprising a modified RuvC domain.
For example, when the type II CRISPR enzyme is a wild-type SpCas9, the nickase may be a SpCas9 mutant in which residue 10 in the amino acid sequence of wild-type SpCas9 is mutated from aspartate to alanine and the nuclease activity of the RuvC domain is inactivated. Here, the produced nickase (i.e., SpCas9 mutant) has nuclease activity of the HNH domain and is thus capable of cleaving the complementary strand of the target gene or nucleic acid, i.e., the strand that forms a complementary binding with the gRNA.
In another example, when the type II CRISPR enzyme is a wild-type CjCas9, the nickase may be a CjCas9 mutant in which residue 8 in the amino acid sequence of wild-type CjCas9 is mutated from aspartic acid to alanine and the nuclease activity of the RuvC domain is inactivated. Here, the resulting nickase (i.e., CjCas9 mutant) has the nuclease activity of the HNH domain and is therefore capable of cleaving the complementary strand of the target gene or nucleic acid, i.e., the strand that forms a complementary binding with the gRNA.
The CRISPR enzyme may be an inactivated CRISPR enzyme.
The term "inactivation" refers to a state of complete loss of wild-type CRISPR enzyme function (i.e., a first function to cleave a first strand of double-stranded DNA, and a second function to cleave a second strand of double-stranded DNA). CRISPR enzymes in this state are referred to as inactivated CRISPR enzymes.
An inactivated CRISPR enzyme can be provided with an inactivated nuclease by mutation in a domain of the wild-type CRISPR enzyme having nuclease activity.
The inactivated CRISPR enzyme may be one in which the nuclease activity of the RuvC domain and the HNH domain is inactivated due to a mutation. That is, the inactivated CRISPR enzyme may not comprise the nuclease activity of the RuvC domain and the HNH domain of the CRISPR enzyme, for which purpose the RuvC domain and the HNH domain may be manipulated or modified.
In one example, when the CRISPR enzyme is a type II CRISPR enzyme, the inactivated CRISPR enzyme may be a type II CRISPR enzyme comprising a modified RuvC domain and an HNH domain.
For example, when the type II CRISPR enzyme is a wild-type SpCas9, the inactivated CRISPR enzyme may be a SpCas9 mutant in which the nuclease activity of the RuvC domain and the HNH domain is inactivated by mutating the 10 th and 840 th residues from aspartic acid and histidine to alanine, respectively, in the amino acid sequence of the wild-type SpCas 9. Here, the generated inactivated CRISPR enzyme (i.e., SpCas9 mutant) has nuclease activity of the inactivated RuvC domain and HNH domain, and thus can not cleave double strands of a target gene or nucleic acid at all.
In another example, when the type II CRISPR enzyme is a wild-type CjCas9, the inactivated CRISPR enzyme may be a CjCas9 mutant in which the nuclease activity of the RuvC domain and the HNH domain is inactivated by mutating the 8 th residue and the 559 th residue from aspartic acid and histidine to alanine, respectively, in the amino acid sequence of the wild-type CjCas 9. Here, the generated inactivated CRISPR enzyme (i.e., SpCas9 mutant) has nuclease activity of the inactivated RuvC domain and HNH domain, and thus can not cleave double strands of a target gene or nucleic acid at all.
In addition to the nuclease activities described above, CRISPR enzymes can have helicase activity, i.e., the ability to unwind the helical structure of a double-stranded nucleic acid.
In addition, the CRISPR enzyme can be modified to have full, incomplete, or partial activity of helicase activity.
The CRISPR enzyme can be a CRISPR enzyme mutant that has been artificially manipulated or modified by a wild-type CRISPR enzyme.
The CRISPR enzyme mutants can be artificially manipulated or modified to modify the function of a wild-type CRISPR enzyme (i.e., a first function to cleave a first strand of double-stranded DNA, and/or a second function to cleave a second strand of double-stranded DNA).
For example, a CRISPR enzyme mutant can be a form that loses a first function of a wild-type CRISPR enzyme function.
Alternatively, the CRISPR enzyme mutant may be in a form that loses a second function of the wild-type CRISPR enzyme function.
For example, the CRISPR enzyme mutant can be a form that loses a function (i.e., a first function and a second function) of a wild-type CRISPR enzyme.
CRISPR enzyme mutants can form gRNA-CRISPR enzyme complexes by interacting with the gRNA.
The CRISPR enzyme mutants can be artificially manipulated or modified to modify the function of the wild-type CRISPR enzyme in interacting with the gRNA.
For example, a CRISPR enzyme mutant can be in a form that has reduced interaction with a gRNA as compared to a wild-type CRISPR enzyme.
Alternatively, the CRISPR enzyme mutant can be in a form that has increased interaction with the gRNA as compared to a wild-type CRISPR enzyme.
For example, a CRISPR enzyme mutant can be a form that has the first function of a wild-type CRISPR enzyme and reduced interaction with a gRNA.
Alternatively, the CRISPR enzyme mutant can be a form that has the first function of a wild-type CRISPR enzyme and increased interaction with the gRNA.
For example, a CRISPR enzyme mutant can be a form that has the second function of a wild-type CRISPR enzyme and reduced interaction with a gRNA.
Alternatively, the CRISPR enzyme mutant can be a form that has the second function of a wild-type CRISPR enzyme and increased interaction with the gRNA.
For example, a CRISPR enzyme mutant can be a form that does not have the first and second functions of a wild-type CRISPR enzyme, but has reduced interaction with a gRNA.
Alternatively, the CRISPR enzyme mutant can be a form that does not have the first and second functions of a wild-type CRISPR enzyme, but has increased interaction with the gRNA.
Here, depending on the strength of the interaction between the gRNA and the CRISPR enzyme mutant, a variety of gRNA-CRISPR enzyme complexes may be formed, and the function of approaching or cleaving the target sequence may be altered depending on the CRISPR enzyme mutant.
For example, a gRNA-CRISPR enzyme complex formed by a CRISPR enzyme mutation that has reduced interaction with the gRNA is only capable of cleaving either a double or single strand of a target sequence when it is in proximity to or positioned to form a fully complementary binding to the gRNA.
The CRISPR enzyme mutant can be a modification of at least one of the amino acids of a wild-type CRISPR enzyme.
In one example, the CRISPR enzyme mutant can be a substitution of at least one of the amino acids of a wild-type CRISPR enzyme.
In another example, the CRISPR enzyme mutant can be a deletion of at least one of the amino acids of a wild-type CRISPR enzyme.
In yet another example, the CRISPR enzyme mutant can have at least one of the amino acids of the wild-type CRISPR enzyme added.
In one example, the CRISPR enzyme mutant can be a substitution, deletion, and/or addition of at least one of the amino acids of a wild-type CRISPR enzyme.
Furthermore, in addition to the original functions of the wild-type CRISPR enzyme (i.e., a first function to cleave a first strand of double-stranded DNA and a second function to cleave a second strand of double-stranded DNA), the CRISPR enzyme mutants can further comprise optional functional domains. Here, the CRISPR enzyme mutants may have additional functions in addition to the original functions of the wild-type CRISPR enzyme.
The functional domain may be a domain having methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity or nucleic acid binding activity, or a tag or reporter gene for isolating and purifying a protein (including a peptide), but the present invention is not limited thereto.
The tags include histidine (His) tag, V5 tag, FLAG tag, influenza Hemagglutinin (HA) tag, Myc tag, VSV-G tag, and thioredoxin (Trx) tag, and the reporter genes include glutathione-S-transferase (GST), horseradish peroxidase (HRP), Chloramphenicol Acetyltransferase (CAT), β -galactosidase, β -glucuronidase, luciferase, autofluorescent proteins (including Green Fluorescent Protein (GFP), HcRed, DsRed, Cyan Fluorescent Protein (CFP), Yellow Fluorescent Protein (YFP), and Blue Fluorescent Protein (BFP)), but the present invention is not limited thereto.
The functional domain may be a deaminase.
For example, an incomplete or partial CRISPR enzyme may additionally comprise a cytidine deaminase as a functional domain. In one exemplary embodiment, a cytidine deaminase, such as apolipoprotein B editing complex 1(APOBEC1), may be added to SpCas9 nickase to generate a fusion protein. The thus formed [ SpCas9 nickase ] - [ APOBEC1] can be used for editing or nucleotide repair from nucleotide C to T or U, or from nucleotide G to a.
In another example, an incomplete or partial CRISPR enzyme may further comprise a cytidine deaminase as a functional domain. In one embodiment, an adenine deaminase (e.g., a TadA variant, ADAR2 variant, ADAT2 variant, etc.) can be added to the SpCas9 nickase, thereby generating a fusion protein. The thus formed [ SpCas9 nickase ] - [ TadA variant ], [ SpCas9 nickase ] - [ ADAR2 variant ] or [ SpCas9 nickase ] - [ ADAT2 variant ] modifies nucleotide a to inosine, and the modified inosine is recognized as nucleotide G by polymerase and essentially exhibits a repair or editing action from nucleotide a to G, and thus can be used for editing from nucleotide a to G, or from nucleotide T to C, or nucleotide repair.
The functional domain may be a nuclear localization sequence or signal (NLS) or a nuclear export sequence or signal (NES).
In one example, the CRISPR enzyme may comprise one or more NLS, where the one or more NLS may be comprised at or near the N-terminus of the CRISPR enzyme, at or near the C-terminus of the enzyme, or a combination thereof NLS may be a NLS sequence derived from a SV40 virus large T antigen having the amino acid sequence PKKKRKV, a NLS from a nucleoplasmic protein (e.g., a bimorph nucleoplasmic protein NLS having the sequence KRPAATKKAGQAKKKK), a C-myc NLS having the amino acid sequence PAAKRVKLD or RQRRNELKRSP, a hRNPA 1M 9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY, a sequence RMRIKNK KDTAELKRVEELVEELKRAKKDKRRNV from the IBB domain of the input protein α, a sequence VSRKRPRPRPRPR 9 NLR and PPKKARED of the protein sequence p53, a sequence PKPOPL 63K III K IV, a sequence VSRK R III K IV, a sequence of a human p53, a sequence of a mouse pKKRK III K IV, a sequence of a mouse antigen derived from a mouse influenza virus, a sequence of a mouse influenza virus and a mouse glucocorticoid receptor subtype antigen of a mouse.
In addition, the CRISPR enzyme mutants may include split-type CRISPR enzymes prepared by dividing the CRISPR enzyme into two or more parts. The term "split" refers to the functional or structural division of a protein, or the random division of a protein into more than two parts.
The split CRISPR enzyme may be an enzyme with full activity, an enzyme with incomplete or partial activity or an inactivated enzyme.
For example, when the CRISPR enzyme is SpCas9, the split SpCas9 can be split into two parts between residue 656 (tyrosine) and residue 657 (threonine) to create split SpCas 9.
The resolving CRISPR enzyme may optionally comprise additional domains, peptides, polypeptides or proteins for reconstruction (reorganization).
Additional domains, peptides, polypeptides, or proteins for reconstitution can be assembled such that the split CRISPR enzyme is identical or similar in structure to the wild-type CRISPR enzyme.
The additional domains, peptides, polypeptides or proteins used for reconstitution can be FRB and FKBP dimerization domains; intein (intein); ERT and VPR domains; or a domain that forms a heterodimer under specific conditions.
For example, the SpCas9 can be split into two parts between residue 713 (serine) and residue 714 (glycine), thereby generating a split SpCas 9. The FRB domain can be linked to one of the two parts and the FKBP domain to the other part. In the resulting split SpCas9, the FRB domain and FKBP domain can form a dimer in the presence of rapamycin, thereby generating a reconstituted CRISPR enzyme.
The CRISPR enzyme or CRISPR enzyme mutant described herein can be a polypeptide, protein, or nucleic acid having a sequence encoding the polypeptide, protein, and can be codon optimized for a subject into which the CRISPR enzyme or CRISPR enzyme mutant is to be introduced.
The term "codon optimization" refers to a modification of a nucleic acid sequence that improves expression in a host cell by replacing at least one codon in the native sequence with a codon that is more or most frequently used in the host cell while maintaining the native amino acid sequence. Various species have a specific preference for a particular codon for a particular amino acid, which codon preference (difference in codon usage between different organisms) is generally associated with the translation efficiency of the mRNA, and is believed to depend on the identity of the codon translated and the availability of a particular tRNA molecule. The dominant tRNA chosen in the cell typically reflects the codon most frequently used in peptide synthesis. Thus, genes can be customized by optimizing gene expression in a given organism based on codon optimization.
The grnas, CRISPR enzymes, or gRNA-CRISPR enzyme complexes disclosed herein can be delivered into or introduced into a subject in various forms.
The explanation about the above subjects is as described above.
In embodiments, the gRNA and/or CRISPR enzyme can be delivered into or introduced into a subject by a vector comprising a nucleic acid sequence encoding the gRNA and/or CRISPR enzyme, respectively.
The vector may comprise a nucleic acid sequence encoding a gRNA and/or CRISPR enzyme.
In one example, the vector can comprise both nucleic acid sequences encoding grnas and CRISPR enzymes.
In another example, the vector can comprise a nucleic acid sequence encoding a gRNA.
For example, the domains contained in the gRNA may be all contained in the vector, or the domains may be separated and contained separately in the vector.
In another example, the vector may comprise a nucleic acid sequence encoding a CRISPR enzyme.
For example, for CRISPR enzymes, the nucleic acid sequence encoding the CRISPR enzyme may be contained entirely within the vector, or it may be detached and contained separately within the vector.
The carrier may comprise one or more regulatory/control components.
Here, the regulating/controlling component may include: promoters, enhancers, introns, polyadenylation signals, Kozak consensus sequences, Internal Ribosome Entry Sites (IRES), splice acceptors, and/or 2A sequences.
The promoter may be a promoter recognized by RNA polymerase II.
The promoter may be a promoter recognized by RNA polymerase III.
The promoter may be an inducible promoter.
The promoter may be a subject-specific promoter.
The promoter may be a viral promoter or a non-viral promoter.
For promoters, suitable promoters can be used depending on the control region (i.e., the nucleic acid sequence encoding the gRNA and/or CRISPR enzyme).
For example, promoters that can be used for gRNAs can be the H1, EF-1a, tRNA, or U6 promoters. For example, a promoter useful for CRISPR enzymes can be a CMV, EF-1a, EFs, MSCV, PGK, or CAG promoter.
The vector may be a viral vector or a recombinant viral vector.
The virus may be a DNA virus or an RNA virus.
Here, the DNA virus may be a double stranded DNA (dsdna) virus or a single stranded DNA (ssdna) virus.
Here, the RNA virus may be a single-stranded RNA (ssrna) virus.
The virus may be, but is not limited to, a retrovirus, lentivirus, adenovirus, adeno-associated virus (AAV), vaccinia virus, poxvirus, or herpes simplex virus.
In one example, a nucleic acid sequence encoding a gRNA and/or CRISPR enzyme can be delivered or introduced by a recombinant lentivirus.
In another example, a nucleic acid sequence encoding a gRNA and/or CRISPR enzyme can be delivered or introduced by a recombinant adenovirus.
In yet another example, a nucleic acid sequence encoding a gRNA and/or CRISPR enzyme can be delivered or introduced by a recombinant AAV.
In yet another example, the nucleic acid sequence encoding the gRNA and/or CRISPR enzyme can be delivered or introduced by a mixed virus (e.g., a mixture of one or more of the viruses listed herein).
In embodiments, a form of a gRNA-CRISPR enzyme complex can be delivered into or introduced into a subject.
For example, the gRNA may be DNA, RNA, or a mixture thereof. CRISPR enzymes can be peptides, polypeptides, or proteins.
In one example, the grnas and CRISPR enzymes can be delivered into or introduced into a subject in the form of a gRNA-CRISPR enzyme complex (i.e., Ribonucleoprotein (RNP)) that includes an RNA-type gRNA and a protein-type CRISPR.
The gRNA-CRISPR enzyme complex can be delivered into or introduced into a subject by electroporation, microinjection, transient cell compression or extrusion (e.g., as described in the literature [ Lee et al (2012) nanolett, 12, 6322-.
The gRNA-CRISPR enzyme complexes disclosed in the present specification can be used for the manual manipulation or modification of target genes (i.e., immunomodulatory genes).
The gRNA-CRISPR enzyme complex (i.e., CRISPR complex) described above can be used to manipulate or modify a target gene. Here, the manipulation or modification of the target gene includes all the following stages: i) cleaving or damaging the target gene; and ii) repair or restoration of the damaged target gene.
i) Cleavage or damage of a target gene can use the CRISPR complex to cleave or damage the target gene, particularly to cleave or damage a target sequence in the target gene.
The target sequence may be a target of a gRNA-CRISPR enzyme complex, and the target sequence may or may not comprise a PAM sequence recognized by the CRISPR enzyme. Such target sequences can provide practitioners with important criteria for designing grnas.
The target sequence is specifically recognized by the gRNA of the gRNA-CRISPR enzyme complex, so that the gRNA-CRISPR enzyme complex can be placed in proximity to the recognized target sequence.
"cleavage" at the target site refers to the cleavage of the polynucleotide covalent backbone. Cleavage can include, but is not limited to, enzymatic or chemical hydrolysis of the phosphodiester bond, and can be performed by a variety of other methods. Both single-stranded and double-stranded cleavage are possible, double-stranded cleavage may occur as a result of two different single-stranded cleavages. Double-stranded cleavage can result in blunt-ended or staggered (staggered) ends.
In one example, cleaving or damaging the target gene using the CRISPR complex can be complete cleaving or damaging of the double strand of the target sequence.
In embodiments, when the CRISPR enzyme is a wild-type SpCas9, the CRISPR complex can completely cleave a double strand of the target sequence that forms a complementary binding with the gRNA.
In another embodiment, when the CRISPR enzymes are SpCas9 nickase (D10A) and SpCas9 nickase (H840A), each CRISPR complex can separately cleave both single strands of the target sequence that form complementary binding to the gRNA. That is, SpCas9 nickase (D10A) cleaves a complementary single strand of the target sequence that forms a complementary binding to the gRNA, while SpCas9 nickase (H840A) cleaves a non-complementary single strand of the target sequence that forms a complementary binding to the gRNA, either sequentially or simultaneously.
In another example, cleaving or damaging the target gene or nucleic acid using the CRISPR complex can be cleaving or damaging only a single strand of the double strand of the target sequence. Here, the single strand may be a guide nucleic acid-binding sequence (i.e., a complementary single strand) that forms complementary binding with the gRNA in the target sequence, or a guide nucleic acid-non-binding sequence (i.e., a single strand that is non-complementary to the gRNA) that does not form complementary binding with the gRNA.
In one embodiment, when the CRISPR enzyme is a SpCas9 nickase (D10A), the guide nucleic acid binding sequence in the CRISPR complex cleavable target sequence that forms a complementary binding to the gRNA, i.e., the SpCas9 nickase (D10A) can cleave the complementary single strand, while the guide nucleic acid non-binding sequence that does not form a complementary binding to the gRNA (i.e., the single strand that is non-complementary to the gRNA) may not be cleaved.
In another embodiment, when the CRISPR enzyme is a SpCas9 nickase (H840A), the CRISPR complex can cleave a guide nucleic acid non-binding sequence in the target sequence that does not form a complementary binding to the gRNA, i.e., the SpCas9 nickase (H840A) can cleave a single strand that is non-complementary to the gRNA, while a guide nucleic acid binding sequence in the target sequence that forms a complementary binding to the gRNA (i.e., a complementary single strand) can be left uncleaved.
In yet another example, cleavage or damage to a target gene or nucleic acid using a CRISPR complex can be a partial removal of a nucleic acid fragment.
In embodiments, when a CRISPR complex is formed from two grnas that form complementary binding to different respective target sequences and a wild-type SpCas9, the double strand of the target sequence that forms complementary binding to the first gRNA may be cleaved and the double strand of the target sequence that forms complementary binding to the second gRNA may be cleaved, thereby deleting the nucleic acid fragment via the first and second grnas and SpCas 9.
ii) with respect to repair or restoration of the damaged target gene, repair or restoration may be performed by non-homologous end joining (NHEJ) or homologous mediated repair (HDR).
Non-homologous end joining (NHEJ) is a method of recovering or repairing a double-stranded break in DNA by ligating both ends of a double-stranded or single-stranded cut, and in general, a damaged double-stranded is repaired when two compatible ends formed by a double-stranded break (e.g., cut) are continuously brought into contact with each other so that the two ends are completely joined. NHEJ is a recovery method that can be used throughout the cell cycle and typically occurs when there is no homologous genome as a template in the cell (e.g. G1 phase).
During repair of damaged genes or nucleic acids using NHEJ, the nucleic acid sequence in the NHEJ repair region undergoes some insertions and/or deletions (indels) that result in a frame shift, producing a frameshifted transcriptome mRNA. As a result, intrinsic function is lost due to nonsense-mediated decay (nonsense-mediated decay) or failure of normal protein synthesis. Furthermore, mutations resulting from a considerable number of insertions or deletions in the sequence can lead to a disruption of the protein function, even if the reading frame remains unchanged. Mutations are locus dependent due to the potential for lower tolerance to mutations in important functional domains than to mutations in non-important regions of the protein.
Since indel mutations produced by NHEJ in the native state cannot be predicted, a particular indel sequence is preferably located in a designated damaged region and may be from a small region of minor homology. Conventionally, deletions range in length from 1bp to 50bp, insertions tend to be shorter, and often comprise short repeated sequences that directly surround the damaged region.
Furthermore, NHEJ is a process that causes mutations, and can be used to delete short sequence motifs when it is not necessary to generate a specific final sequence.
This NHEJ can be used for specific knockdown of genes targeted by the CRISPR complex. A CRISPR enzyme (e.g., Cas9 or Cpf1) can be used to cleave a double strand or two single strands of a target gene or nucleic acid and can induce a specific knockout of the target gene or nucleic acid by NHEJ to have an indel of the damaged double strand or two single strands in the target gene or nucleic acid. Here, the site of the target gene or nucleic acid cleaved by the CRISPR enzyme may be in a non-coding region or a coding region; in addition, the target gene or nucleic acid recovered by NHEJ may be at a site that is non-coding or coding.
In one example, various insertions and deletions (indels) can occur in the restored region due to the process of cleaving the double strand of the target gene by using the CRISPR complex and restoring through NHEJ.
The term "indels" collectively refers to such mutations: in which some nucleotides are inserted or deleted in the nucleotide sequence of the DNA. As described above, when the nucleic acid-editing protein complex is directed to cleave a nucleic acid (DNA, RNA) of an immunomodulatory gene, an indel may be an indel that introduces the target sequence during repair by homologous recombination (HDR) or non-homologous end joining (NHEJ) mechanisms.
Homology Directed Repair (HDR) is an error-free correction method that repairs or restores damaged genes or nucleic acids using a homologous sequence as a template, and in general, repairs or restores damaged DNA (i.e., restores intrinsic information of cells) using information of a complementary nucleotide sequence that is not modified or information of sister chromatids. The most common type of HDR is Homologous Recombination (HR). HDR is a repair or restoration process that usually occurs in the S phase or G2/M phase of actively dividing cells.
To repair or restore damaged DNA via HDR without using sister chromatids or complementary nucleotide sequences of the cell, a DNA template artificially synthesized using information of complementary nucleotide sequences or homologous nucleotide sequences (i.e., a nucleic acid template comprising complementary nucleotide sequences or homologous nucleotide sequences) may be provided to the cell to repair or restore damaged DNA. Here, when a nucleic acid sequence or a nucleic acid fragment is further added to the nucleic acid template to repair the damaged DNA, the nucleic acid sequence or the nucleic acid fragment further added to the damaged DNA may be knocked in. The further added nucleic acid sequence or nucleic acid fragment may be a nucleic acid sequence or nucleic acid fragment modified from a target gene or nucleic acid modified by mutation of a normal gene or nucleic acid, or a gene or nucleic acid desired to be expressed in a cell, but is not limited thereto.
In one example, a CRISPR complex can be used to cleave a double or single strand of a target gene or nucleic acid, and a nucleic acid template (which comprises a nucleotide sequence complementary to a nucleotide sequence adjacent to the cleavage site) can be provided to a cell to repair or restore the cleaved nucleotide sequence in the target gene or nucleic acid by HDR methods.
Here, the nucleic acid template comprising the complementary nucleotide sequence may have damaged DNA (i.e., double strand or single strand cleaved in the complementary nucleotide sequence), and further comprise a nucleic acid sequence or a nucleic acid fragment desired to be inserted into the damaged DNA. Additional nucleic acid sequences or nucleic acid fragments can be inserted into the damaged DNA (i.e., the cleavage site of the target gene or nucleic acid) using a nucleic acid template comprising a complementary base sequence and the nucleic acid sequence or nucleic acid fragment to be inserted. Here, the nucleic acid sequence or nucleic acid fragment to be inserted and the additional nucleic acid sequence or nucleic acid fragment may be a nucleic acid sequence or nucleic acid fragment that modifies a target gene or nucleic acid that is modified by mutation of a normal gene or nucleic acid, or a gene or nucleic acid to be expressed in a cell. The complementary nucleotide sequence may be a nucleotide sequence that forms a complementary binding with the damaged DNA (i.e., a nucleotide sequence to the left or right of a double strand or a single strand in which the target gene or nucleic acid is cleaved). Alternatively, the complementary nucleotide sequence may be a nucleotide sequence that forms a complementary bond with the damaged DNA (i.e., the 3 'and 5' ends of the double or single strand where the target gene or nucleic acid is cleaved). The complementary nucleotide sequence may be 15bp to 3000bp, and the length or size of the complementary nucleotide sequence may be designed appropriately according to the size of the nucleic acid template or the target gene or the nucleic acid. Here, as the nucleic acid template, a double-stranded or single-stranded nucleic acid may be used, or it may be linear or circular, but the present invention is not limited thereto.
In another example, a CRISPR complex can be used to cleave a double-stranded or single-stranded target gene or nucleic acid, a nucleic acid template (which comprises a homologous nucleotide sequence of nucleotide sequences proximal to the cleavage site) can be provided to a cell, and the cleaved nucleotide sequence in the target gene or nucleic acid can be repaired or restored by HDR methods.
Here, the nucleic acid template comprising the homologous nucleotide sequence may have damaged DNA (i.e., a double-stranded or single-stranded homologous nucleotide sequence that is cleaved), and further comprise a nucleic acid sequence or a nucleic acid fragment desired to be inserted into the damaged DNA. Additional nucleic acid sequences or nucleic acid fragments can be inserted into the damaged DNA (i.e., the cleavage site of the target gene or nucleic acid) using a nucleic acid template comprising the homologous base sequence and the nucleic acid sequence or nucleic acid fragment to be inserted. Here, the nucleic acid sequence or nucleic acid fragment to be inserted and the additional nucleic acid sequence or nucleic acid fragment may be a nucleic acid sequence or nucleic acid fragment that modifies a target gene or nucleic acid that is modified by mutation of a normal gene or nucleic acid, or a gene or nucleic acid to be expressed in a cell. The homologous nucleotide sequence may be a nucleotide sequence having homology with the damaged DNA, that is, a nucleotide sequence having homology with the left and right nucleotide sequences of the double strand or single strand cleaved in the target gene or nucleic acid. Alternatively, the homologous nucleotide sequence may be a nucleotide sequence having homology with the damaged DNA, that is, a base sequence having homology with 3 'and 5' ends of the double strand or single strand cleaved in the target gene or nucleic acid. The homologous nucleotide sequence may be a 15bp-3000bp nucleotide sequence, and the length or size of the homologous nucleotide sequence may be appropriately designed according to the size of the nucleic acid template or the target gene or nucleic acid. Here, as the nucleic acid template, a double-stranded or single-stranded nucleic acid may be used, or it may be linear or circular, but the present invention is not limited thereto.
In addition to NHEJ and HDR, there are methods of repair or restoration of damaged target genes. For example, the method of repair or restoration of a damaged target gene may be single strand annealing, single strand break repair, mismatch repair, or nucleotide damage repair or a method using nucleotide damage repair.
Single-strand annealing (SSA) is a method of repairing double-stranded breaks between two repetitive sequences present in a target nucleic acid, typically using repetitive sequences of more than 30bp nucleotide sequence. The repetitive sequences can be cleaved (to create sticky ends) to create single strands at each break end of the target nucleic acid duplex; furthermore, single-chain overhangs (overhans) containing the repeats are coated with RPA protein after cleavage to prevent improper annealing of the repeats to each other. RAD52 binds to each repeat on the overhang and aligns sequences capable of annealing to complementary repeats. After annealing, the single-stranded overhang (flap) of the overhang is cleaved, and new DNA is synthesized to fill in a specific gap, thereby restoring the DNA double strand. The result of this repair is that the DNA sequence between the two repeats is deleted, the length of which can depend on a number of factors (including the position of the two repeats and the path or progression of the cut as used herein).
For modification or correction of a target nucleic acid sequence, SSA uses complementary sequences (i.e., complementary repeat sequences) similar to HDR; unlike HDR, SSA does not require a nucleic acid template.
Single Strand Break Repair (SSBR) can repair single strand breaks in the genome by a different mechanism than that described above. In the case of single-stranded DNA breaks, PARP1 and/or PARP2 recognize the break and mobilize the repair mechanism. The binding and activity of PARP1 to DNA breaks is transient, promoting SSBR by promoting the stability of the SSBR protein complex in the damaged region. The most important protein in the SSBR complex is XRCC1, which interacts with proteins that facilitate processing of the 3 'and 5' ends of DNA to stabilize the DNA. End-processing typically involves repairing the damaged 3 'end to a hydroxylated state and/or the damaged 5' end to have a phosphate moiety, and DNA gap-filling occurs after end-processing. There are two DNA gap filling methods, short patch (patch) repair, which involves the insertion of a translocated single nucleotide, and long patch repair. After DNA gap filling, DNA ligase facilitates end ligation.
Mismatch Repair (MMR) can act on mismatched DNA nucleotides. The MSH2/6 or MSH2/3 complexes each have ATPase activity and thus play an important role in recognition of mismatches and priming repair, and MSH2/6 mainly recognizes nucleotide-nucleotide mismatches and recognizes mismatches of one or two nucleotides, whereas MSH2/3 mainly recognizes longer mismatches.
Base Excision Repair (BER) is a repair method active throughout the cell cycle that is used to remove small, non-helically twisted nucleotide damage regions from the genome. In damaged DNA, damaged nucleotides are removed by cleavage of the N-glycosidic bond connecting the base to the deoxyribose-phosphate backbone, followed by cleavage of the phosphodiester backbone, resulting in single-stranded DNA breaks. The damaged single-stranded end thus formed is removed and the gap caused by the single-stranded removal is filled with a new complementary base, and then the end of the newly filled complementary base is ligated to the backbone using a DNA ligase, resulting in repair or restoration of the damaged DNA.
NER (nucleotide excision repair) is an important excision mechanism for removing large helically twisted lesions from DNA, and when a lesion is recognized, a short single-stranded DNA fragment containing the damaged region is removed, resulting in a single-stranded gap of 22bp-30bp nucleotide sequence. And filling the generated gap with the new complementary base, and connecting the tail end of the newly filled complementary base to the skeleton by using DNA ligase to repair or recover the damaged DNA.
The effects of manual manipulation of target genes (i.e., immunoregulatory genes) with the gRNA-CRISPR complex can be, to a large extent, knockdown, knockin, and knock-out.
The term "knock-out" refers to inactivation of a target gene or nucleic acid, whereas "inactivation of a target gene or nucleic acid" refers to a state in which transcription and/or translation of the target gene or nucleic acid does not occur. The transcription and translation of a gene causing a disease or a gene having an abnormal function can be inhibited by knockout, and the expression of a protein can be prevented.
For example, when the target gene or chromosome is edited or corrected using the gRNA-CRISPR enzyme complex (i.e., CRISPR complex), the target gene or chromosome may be cleaved using the CRISPR complex. The CRISPR complex can be used to repair damaged target genes or chromosomes via NHEJ. Due to NHEJ, the damaged target gene or chromosome may have an indel, so that a specific knockout for the target gene or chromosome may be induced.
In another example, when a target gene or chromosome is edited or modified using a gRNA-CRISPR enzyme complex (i.e., CRISPR complex) and a donor, the target gene or nucleic acid can be cleaved using the CRISPR complex. Target genes or nucleic acids damaged by CRISPR complexes can be restored with HDR using a donor. Here, the donor contains a complementary nucleotide sequence and a nucleotide sequence desired to be inserted. Here, the number of nucleotide sequences desired to be inserted may be adjusted depending on the position or purpose of the insertion. When a damaged gene or chromosome is repaired by using a donor, a desired inserted nucleotide sequence is inserted into the damaged nucleotide sequence region, so that specific knockout of the target gene or chromosome can be induced.
The term "knockdown" refers to a decrease in transcription and/or translation of a target gene or nucleic acid or expression of a target protein. Modulation of gene or protein overexpression by knockdown can prevent morbidity or treat disease.
For example, when a target gene or chromosome is edited or corrected using a gRNA-CRISPR inactivator-transcription repression active domain complex (i.e., a CRISPR inactivating complex comprising a transcription repression active domain), the CRISPR inactivating complex can specifically bind to the target gene or chromosome, and transcription of the target gene or chromosome can be repressed by the transcription repression active domain contained in the CRISPR inactivating complex, thereby inducing knockdown (in which expression of the corresponding gene or chromosome is repressed).
In another example, when the gRNA-CRISPR enzyme complex (i.e., CRISPR complex) is used to edit or modify a target gene or chromosome, the CRISPR complex can cleave the promoter and/or enhancer region of the target gene or chromosome. Here, the gRNA may recognize a partial nucleotide sequence as a target sequence in a promoter and/or enhancer region of a target gene or chromosome. Target genes or chromosomes damaged by CRISPR complexes can be restored by NHEJ. Due to NHEJ, the damaged target gene or chromosome may have an indel, so that a specific knockout for the target gene or chromosome may be induced. Alternatively, when a donor is selectively used, the target gene or chromosome damaged by the CRISPR complex can be restored by HDR. When the damaged gene or chromosome is restored using a donor, a desired inserted nucleotide sequence is inserted into the damaged nucleotide sequence region, so that specific knockdown for the target gene or chromosome can be induced.
The term "knock-in" refers to the insertion of a particular nucleic acid or gene into a target gene or nucleic acid, and in particular, the term "particular nucleic acid or gene" refers to a nucleic acid or gene that is intended to be inserted or desired to be expressed. Knock-in can be used for the treatment of disease by making precise corrections to the mutant gene causing the disease or by inserting a normal gene to induce normal gene expression.
Furthermore, the knock-in may require an additional donor.
For example, when a target gene or nucleic acid is edited or modified using a gRNA-CRISPR enzyme complex (i.e., CRISPR complex) and a donor, the target gene or nucleic acid can be cleaved using the CRISPR complex. The CRISPR complex can be used to restore damaged target genes or nucleic acids by HDR. Here, the donor contains a specific nucleic acid or gene, and the specific nucleic acid or gene can be inserted into the damaged gene or chromosome using the donor. Here, the inserted specific nucleic acid or gene may induce protein expression.
As embodiments disclosed herein, the gRNA-CRISPR enzyme complex can be manually manipulated or modified for the following genes: PD-1 gene, CTLA-4 gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene, PPP2r2d gene, TET2 gene, PSGL-1 gene, A20 gene and/or KDM6A gene.
The gRNA-CRISPR enzyme complex can specifically recognize the target sequences of the following genes: PD-1 gene, CTLA-4 gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene, PPP2r2d gene, TET2 gene, PSGL-1 gene, A20 gene and/or KDM6A gene.
The gRNA of the gRNA-CRISPR enzyme complex can specifically recognize a target sequence, thereby positioning the gRNA-CRISPR enzyme complex in proximity to the recognized target sequence.
The target sequence may be a region or a range in which the PD-1 gene, CTLA-4 gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene, PPP2r2d gene, TET2 gene, PSGL-1 gene, A20 gene and/or KDM6A gene are artificially modified.
The target sequence may be a contiguous 10bp-25bp nucleotide sequence located in the promoter region of the following genes: PD-1 gene, CTLA-4 gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene, PPP2r2d gene, TET2 gene, PSGL-1 gene, A20 gene and/or KDM6A gene.
The target sequence may be a contiguous 10bp-25bp nucleotide sequence located in the intronic region of the following genes: PD-1 gene, CTLA-4 gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene, PPP2r2d gene, TET2 gene, PSGL-1 gene, A20 gene and/or KDM6A gene.
The target sequence may be a contiguous 10bp-25bp nucleotide sequence located in the exon regions of the following genes: PD-1 gene, CTLA-4 gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene, PPP2r2d gene, TET2 gene, PSGL-1 gene, A20 gene and/or KDM6A gene.
The target sequence may be a contiguous 10bp-25bp nucleotide sequence located in the enhancer region of the following genes: PD-1 gene, CTLA-4 gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene, PPP2r2d gene, TET2 gene, PSGL-1 gene, A20 gene and/or KDM6A gene.
The target sequence may be a contiguous 10bp-25bp nucleotide sequence located in the 3' -UTR region of the following genes: PD-1 gene, CTLA-4 gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene, PPP2r2d gene, TET2 gene, PSGL-1 gene, A20 gene and/or KDM6A gene.
The target sequence may be a contiguous 10bp-25bp nucleotide sequence located in the 5' -UTR region of the following genes: PD-1 gene, CTLA-4 gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene, PPP2r2d gene, TET2 gene, PSGL-1 gene, A20 gene and/or KDM6A gene.
The target sequence may be a contiguous 10bp-25bp nucleotide sequence adjacent to the 5 'end and/or 3' end region of a motif (PAM) sequence in the nucleotide sequence of the following gene: PD-1 gene, CTLA-4 gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene, PPP2r2d gene, TET2 gene, PSGL-1 gene, A20 gene and/or KDM6A gene.
Here, the PAM sequence may be, for example, one or more of the following (described in the 5 'to 3' direction)
NGG (N is A, T, C or G);
NNNNRYACs (each N is independently A, T, C or G; R is A or G; Y is C or T);
NNAGAAW (N is each independently A, T, C or G; W is A or T);
NNNNGATT (N is each independently A, T, C or G);
NNGRR (T) (N is A, T, C or G independently; R is A or G; Y is C or T); and
TTN (N is A, T, C or G).
In embodiments, the target sequence may be one or more nucleotide sequences selected from the nucleotide sequences set forth in table 1.
A gRNA-CRISPR enzyme complex can be formed from the gRNA and CRISPR enzyme.
A gRNA may comprise a guide domain capable of forming a partial or complete complementary binding with a guide nucleic acid binding sequence in the target sequence of the following gene: PD-1 gene, CTLA-4 gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene, PPP2r2d gene, TET2 gene, PSGL-1 gene, A20 gene and/or KDM6A gene.
The guide domain may be a nucleotide sequence that is complementary to the guide nucleic acid binding sequence, e.g., having at least 70%, 75%, 80%, 85%, 90%, or 95% or more complementarity or complete complementarity.
The guide domain may comprise a nucleotide sequence complementary to a guide nucleic acid binding sequence in a target sequence of the PD-1 gene. Here, the complementary nucleotide sequence may comprise 0-5, 0-4, 0-3, or 0-2 mismatches.
The guide domain may comprise a nucleotide sequence complementary to a guide nucleic acid binding sequence in a target sequence of the CTLA-4 gene. Here, the complementary nucleotide sequence may comprise 0-5, 0-4, 0-3, or 0-2 mismatches.
The guide domain may comprise a nucleotide sequence complementary to a guide nucleic acid binding sequence in the target sequence of the a20 gene. Here, the complementary nucleotide sequence may comprise 0-5, 0-4, 0-3, or 0-2 mismatches.
The guide domain may comprise a nucleotide sequence complementary to a guide nucleic acid binding sequence in a target sequence of the DGKA gene. Here, the complementary nucleotide sequence may comprise 0-5, 0-4, 0-3, or 0-2 mismatches.
The guide domain may comprise a nucleotide sequence complementary to a guide nucleic acid binding sequence in a target sequence of the DGKZ gene. Here, the complementary nucleotide sequence may comprise 0-5, 0-4, 0-3, or 0-2 mismatches.
The leader domain may comprise a nucleotide sequence that is complementary to a leader nucleic acid binding sequence in the target sequence of the FAS gene. Here, the complementary nucleotide sequence may comprise 0-5, 0-4, 0-3, or 0-2 mismatches.
The guide domain may comprise a nucleotide sequence complementary to a guide nucleic acid binding sequence in a target sequence of the EGR2 gene. Here, the complementary nucleotide sequence may comprise 0-5, 0-4, 0-3, or 0-2 mismatches.
The targeting domain may comprise a nucleotide sequence complementary to a targeting nucleic acid binding sequence in the target sequence of the PPP2r2d gene. Here, the complementary nucleotide sequence may comprise 0-5, 0-4, 0-3, or 0-2 mismatches.
The targeting domain may comprise a nucleotide sequence complementary to a targeting nucleic acid binding sequence in the target sequence of TET2 gene. Here, the complementary nucleotide sequence may comprise 0-5, 0-4, 0-3, or 0-2 mismatches.
The guide domain may comprise a nucleotide sequence complementary to a guide nucleic acid binding sequence in a target sequence of the PSGL-1 gene. Here, the complementary nucleotide sequence may comprise 0-5, 0-4, 0-3, or 0-2 mismatches.
The leader domain may comprise a nucleotide sequence complementary to a leader nucleic acid binding sequence in the target sequence of the KDM6A gene. Here, the complementary nucleotide sequence may comprise 0-5, 0-4, 0-3, or 0-2 mismatches.
The gRNA may comprise one or more domains selected from the group consisting of a first complementary domain, a linking domain, a second complementary domain, a proximal domain, and a tail domain.
The CRISPR enzyme may be one or more proteins selected from the group consisting of: a Cas9 protein derived from streptococcus pyogenes, a Cas9 protein derived from campylobacter jejuni, a Cas9 protein derived from streptococcus thermophilus, a Cas9 protein derived from staphylococcus aureus, a Cas9 protein derived from neisseria meningitidis, and a Cpf1 protein. In one example, the editing protein can be a campylobacter jejuni-derived Cas9 protein or a staphylococcus aureus-derived Cas9 protein.
Depending on the type of gRNA and CRISPR enzyme, the gRNA-CRISPR enzyme complex can manually manipulate or modify the following genes: PD-1 gene, CTLA-4 gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene, PPP2r2d gene, TET2 gene, PSGL-1 gene, A20 gene and/or KDM6A gene.
In one example, when the CRISPR enzyme is SpCas9 protein, consecutive 1bp-50bp, 1bp-40bp, 1bp-30bp, preferably 1bp-25bp nucleotide sequence regions in positions adjacent to the 5 'end and/or 3' end of a 5'-NGG-3' (N is A, T, G or C) PAM sequence present in a target region of a manually manipulated or modified gene, which is a PD-1 gene, CTLA-4 gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene, PPP2r2d gene, TET2 gene, PSGL-1 gene, a20 gene and/or KDM6A gene, may comprise one or more modifications in the region of the nucleotide sequence that are manually manipulated or modified:
i) a deletion of one or more nucleotides;
ii) replacing one or more nucleotides with a nucleotide different from the wild-type gene;
iii) insertion of one or more nucleotides; or
iv) a combination of two or more selected from the above i) -iii).
In another example, when the CRISPR enzyme is a CjCas9 protein, the contiguous 1bp-50bp, 1bp-40bp, 1bp-30bp, preferably 1bp-25bp nucleotide sequence region in the position adjacent to the 5 'end and/or 3' end of the PAM sequence of 5 '-nnnrryac-3' (N is each independently A, T, G or C; R is a or G; Y is C or T) present in the target region of the artificially manipulated or modified gene, which is a PD-1 gene, CTLA-4 gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene, PPP2R2d gene, TET2 gene, PSGL-1 gene, a20 gene, and/or KDM6A gene:
i) a deletion of one or more nucleotides;
ii) replacing one or more nucleotides with a nucleotide different from the wild-type gene;
iii) insertion of one or more nucleotides; or
iv) a combination of two or more selected from the above i) -iii).
In yet another example, when the CRISPR enzyme is a StCas9 protein, consecutive 1bp-50bp, 1bp-40bp, 1bp-30bp, preferably 1bp-25bp nucleotide sequence regions in the positions adjacent to the 5 'end and/or 3' end of the 5'-NNAGAAW-3' (N is each independently A, T, G or C; W is a or T) PAM sequence present in the target region of the artificially manipulated or modified gene may comprise one or more of the following modifications in the region of the nucleotide sequence, because of the artificially manipulated or modified PD-1 gene, CTLA-4 gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene, PPP2r2d gene, TET2 gene, PSGL-1 gene, a20 gene and/or KDM6A gene:
i) a deletion of one or more nucleotides;
ii) replacing one or more nucleotides with a nucleotide different from the wild-type gene;
iii) insertion of one or more nucleotides; or
iv) a combination of two or more selected from the above i) -iii).
In one example, when the CRISPR enzyme is NmCas9 protein, the contiguous 1bp-50bp, 1bp-40bp, 1bp-30bp, preferably 1bp-25bp nucleotide sequence region in a position adjacent to the 5 'end and/or 3' end of a5 '-nnnnnngatt-3' (N is each independently A, T, G or C) PAM sequence present in a target region of a manually manipulated or modified gene, which is a PD-1 gene, CTLA-4 gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene, PPP2r2d gene, TET2 gene, PSGL-1 gene, a20 gene, and/or KDM6A gene that is manually manipulated or modified, may comprise one or more modifications in the region of the nucleotide sequence:
i) a deletion of one or more nucleotides;
ii) replacing one or more nucleotides with a nucleotide different from the wild-type gene;
iii) insertion of one or more nucleotides; or
iv) a combination of two or more selected from the above i) -iii).
In another example, when the CRISPR enzyme is a SacAS9 protein, one or more of the following modifications may be included in the contiguous 1bp-50bp, 1bp-40bp, 1bp-30bp, preferably 1bp-25bp nucleotide sequence region in the position adjacent to the 5 'end and/or 3' end of the PAM sequence(s) (N is each independently A, T, G or C; R is A or G; and T is any sequence that may optionally be included) that is present in the target region of the artificially manipulated or modified gene, which is a PD-1 gene, CTLA-4 gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene, PPP2R2d gene, TET2 gene, PSGL-1 gene, A20 gene, and/or KDM6A gene:
i) a deletion of one or more nucleotides;
ii) replacing one or more nucleotides with a nucleotide different from the wild-type gene;
iii) insertion of one or more nucleotides; or
iv) a combination of two or more selected from the above i) -iii).
In another example, when the CRISPR enzyme is a Cpf1 protein, consecutive 1bp-50bp, 1bp-40bp, 1bp-30bp, preferably 1bp-25bp nucleotide sequence regions in positions adjacent to the 5 'end and/or 3' end of a 5'-TTN-3' (N are each independently A, T, G or C) PAM sequence present in a target region of a manually manipulated or modified gene, which is a manually manipulated or modified PD-1 gene, CTLA-4 gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene, PPP2r2d gene, TET2 gene, PSGL-1 gene, a20 gene and/or KDM6A gene, may comprise one or more modifications in the region of the nucleotide sequence:
i) a deletion of one or more nucleotides;
ii) replacing one or more nucleotides with a nucleotide different from the wild-type gene;
iii) insertion of one or more nucleotides; or
iv) a combination of two or more selected from the above i) -iii).
The effect of manual manipulation of the PD-1 gene, CTLA-4 gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene, PPP2r2d gene, TET2 gene, PSGL-1 gene, A20 gene and/or KDM6A gene by means of the gRNA-CRISPR enzyme complex may be a knock-out.
The expression of proteins encoded by the PD-1 gene, CTLA-4 gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene, PPP2r2d gene, TET2 gene, PSGL-1 gene, A20 gene and/or KDM6A gene can be suppressed by the action of manual manipulation of the PD-1 gene, CTLA-4 gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene, PPP2r2d gene, TET2 gene, PSGL-1 gene, A20 gene and/or KDM6A gene by means of the gRNA-CRISPR enzyme complex.
The effect of manual manipulation of the PD-1 gene, CTLA-4 gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene, PPP2r2d gene, TET2 gene, PSGL-1 gene, A20 gene and/or KDM6A gene by means of the gRNA-CRISPR enzyme complex may be a knock-down.
The expression of proteins encoded by the PD-1 gene, CTLA-4 gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene, PPP2r2d gene, TET2 gene, PSGL-1 gene, A20 gene and/or KDM6A gene, respectively, can be reduced by the action of manual manipulation of the PD-1 gene, CTLA-4 gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene, PPP2r2d gene, TET2 gene, PSGL-1 gene, A20 gene and/or KDM6A gene by means of the gRNA-CRISPR enzyme complex.
The effect of manual manipulation of the PD-1 gene, CTLA-4 gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene, PPP2r2d gene, TET2 gene, PSGL-1 gene, A20 gene and/or KDM6A gene by means of the gRNA-CRISPR enzyme complex may be knock-in.
Here, the knock-in effect can be induced by the gRNA-CRISPR enzyme complex (and additionally by the donor comprising the foreign nucleotide sequence or gene).
The expression of peptides or proteins encoded by foreign nucleotide sequences or genes can be achieved by the action of manual manipulation of the PD-1 gene, CTLA-4 gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene, PPP2r2d gene, TET2 gene, PSGL-1 gene, A20 gene and/or KDM6A gene by means of the gRNA-CRISPR enzyme complex.
One aspect of the disclosure relates to a manipulated immune cell.
An "immune cell" is a cell involved in an immune response, which includes all cells involved directly or indirectly in an immune response as well as their pre-differentiated cells.
Immune cells may have functions of cytokine secretion, differentiation into other immune cells, and cytotoxicity. Immune cells also include cells that undergo mutation from their natural state.
The immune cells are differentiated from hematopoietic stem cells in bone marrow, and mainly comprise lymphoid progenitor cells and myeloid progenitor cells; all the following cells were also included: t cells and B cells differentiated from lymphoid progenitor cells and responsible for acquired immunity; and macrophages, eosinophils, neutrophils, basophils, megakaryocytes, erythrocytes, etc., differentiated from myeloid progenitor cells.
Specifically, the cell may be at least one selected from the group consisting of: t cells, e.g. CD8+T cells (e.g. CD 8)+Naive T cells, CD8+Effector T cells, central memory T cells, or effector memory T cells), CD4+T cells, natural killer T cells (NKT cells), regulatory T cells (tregs), stem cell memory T cells; lymphoid progenitor cells; hematopoietic stem cells; natural killer cells (NK cells); a dendritic cell; cytokine-induced killer Cells (CIK); peripheral bloodMonocytes (PBMCs); (ii) a monocyte; macrophages; natural killer t (nkt) cells, and the like.
"manipulated immune cells" refers to immune cells that have undergone artificial manipulation and are not in a natural state. Recently, techniques for enhancing immunity by extracting immune cells from the body and performing manual manipulation have been actively studied. Such manipulated immune cells have proven to be a new therapeutic approach due to the superior immunopotency against certain diseases. In particular, research on the manipulated immune cells has been actively conducted in association with cancer treatment.
The manipulated immune cells can be immune cells that are manually manipulated or modified by a composition for immune cell manipulation. Herein, the term "composition for immune cell manipulation" refers to one or more substances (e.g., DNA, RNA, nucleic acids, proteins, viruses, compositions) used for artificial modification or manipulation of immune cells, e.g., a composition for immune cell manipulation may comprise a part or all of a composition for gene manipulation and may further comprise a nucleic acid encoding a foreign protein for expression of the foreign protein.
The manipulated immune cells may be immune cells produced by gene manipulation.
Here, the gene manipulation may be performed in consideration of the regulation process of gene expression.
In one example, genetic manipulation can be performed in the following steps by selecting a manipulation method suitable for each step: transcriptional regulation, RNA processing regulation, RNA transport regulation, RNA degradation regulation, translational regulation, or protein modification regulation.
For example, gene manipulation can control the expression of genetic information by preventing mRNA using RNA interference (RNAi) or RNA silencing; also, in some cases, the delivery of protein synthesis information during intermediate steps can be prevented by disruption, thereby controlling the expression of genetic information.
In another example, genetic manipulation may use a wild-type enzyme or variant enzyme capable of catalyzing hydrolysis (cleavage) of a DNA or RNA molecule, preferably capable of catalyzing hydrolysis (cleavage) of a bond between nucleic acids in a DNA molecule. A guide nucleic acid-editing protein complex may be used.
For example, gene manipulation may control the expression of genetic information by manipulating a gene using one or more selected from the group consisting of: meganucleases, zinc finger nucleases, CRISPR/Cas9(Cas9 protein), CRISPR-Cpf1(Cpf1 protein), and TALE nucleases.
In a preferred example, without limitation, gene manipulation may be performed by a guide nucleic acid-editing protein complex, as explained above with respect to the guide nucleic acid-editing protein.
Furthermore, the manipulated immune cells may be immune cells having a modified function due to loss or damage of function of a specific protein.
Here, the function of a particular protein may be lost or impaired by the compound.
The compounds can bind to specific proteins and block the function of immunomodulatory factors.
In addition, the compounds may bind to specific proteins and modify the structure of the immune modulatory factors, thereby preventing their normal function.
Alternatively, the function of a particular protein may be lost or impaired by modification of the binding of the protein to the particular protein.
The manipulated immune cells may be functionally manipulated immune cells or hybrid manipulated immune cells.
As an embodiment of the present disclosure, the manipulated immune cells may be functionally manipulated immune cells.
The term "functionally manipulated immune cell" refers to an immune cell in which the natural expression of a wild-type immune modulator has been modified or artificially manipulated to impair the function of the immune modulator.
The term "immunomodulatory factor" refers to a polypeptide or protein encoded by an immunomodulatory gene, and may also be referred to as an immunomodulatory protein that is transcribed, translated, and expressed by an immunomodulatory gene.
The functionally manipulated immune cells may be immune cells that are manipulated to suppress or inhibit the expression of an immunomodulatory factor.
Here, the immune cell that is functionally manipulated may be an immune cell in which an immune regulatory gene is manipulated to repress or inhibit the expression of an immune regulatory factor.
The functionally manipulated immune cells may be immune cells in which an immune cell activity regulating gene is manipulated.
Here, the immune cell that is functionally manipulated may be an immune cell in which one or more genes selected from the group consisting of SHP-1, PD-1, CTLA-4, CBLB, ILT-2, KIR2DL4, and PSGL-1 are inactivated.
The functionally manipulated immune cells may be immune cells in which immune cell growth regulatory genes are manipulated.
Here, the immune cell that is functionally manipulated may be one in which one or more genes selected from DGK- α, DGK-zeta, FAS, EGR2, EGR3, PPP2r2d, and A20 are inactivated in a preferred embodiment, one or more genes selected from DGK- α, DGK-zeta, EGR2, PPP2r2d, and A20 are inactivated.
The functionally manipulated immune cell may be an immune cell in which an immune cell death regulatory gene is manipulated.
Here, the immune cell that is functionally manipulated may be an immune cell in which one or more genes selected from DAXX, BIM, BID, BAD, PD-1, and CTLA-4 are inactivated.
Furthermore, the immune cell that is functionally manipulated may be an immune cell into which an element that induces self-death is inserted.
The functionally manipulated immune cells may be immune cells in which an immune cell depleting regulatory element is manipulated.
Here, the immune cell that is functionally manipulated may be an immune cell in which one or more genes selected from TET2, WNT, and AKT are inactivated.
A functionally manipulated immune cell may be one in which a cytokine secretion element is manipulated.
A functionally manipulated immune cell may be one in which the antigen binding regulatory element is manipulated.
Here, the immune cell that is functionally manipulated may be an immune cell in which one or more genes selected from dCK, CD52, B2M, and MHC are inactivated.
The immune cell that is functionally manipulated may be an immune cell in which an immunomodulatory gene different from the aforementioned gene is manipulated.
The functionally manipulated immune cells may be immune cells in which one or more immunomodulatory genes are manipulated simultaneously. Here, one or more immunomodulatory genes may be manipulated.
Here, when an immune regulatory gene is manipulated, a new immune efficacy is not necessarily exhibited. Manipulation of an immunomodulatory gene can result in or inhibit a variety of novel immune potencies.
The functionally manipulated immune cell may be an immune cell in which a gene encoding a wild-type receptor other than an immunomodulatory gene is manipulated.
Here, the wild-type receptor may be a T Cell Receptor (TCR).
The functionally manipulated immune cells may be those in which wild type receptors are absent or are present at a lower rate on the surface.
The functionally manipulated immune cells may be immune cells in which wild-type receptors are present in large proportion on the surface.
The functionally manipulated immune cell may be one in which the wild-type receptor has enhanced recognition of a particular antigen.
By manipulating wild-type receptors and immunomodulatory genes, immune cells that are functionally manipulated can have novel immunological potency.
The new immunogenic efficacy may be an immunogenic efficacy in which the ability to recognize a specific antigen is modulated.
The new immunogenic efficacy may be an immunogenic efficacy in which the ability to recognize a specific antigen is improved.
In particular, the specific antigen may be a disease antigen, such as a cancer cell antigen.
The new immunogenic efficacy may be an immunogenic efficacy in which the ability to recognize a specific antigen is deteriorated.
The new immunopotency can be an immunopotency in which the new immunopotency is improved.
The new immunogenic effect may be an immunogenic effect in which immune cell growth is modulated. In particular, the immunopotency may be one in which growth and differentiation are promoted or delayed.
The new immunopotency may be an immunopotency that modulates immune cell death. In particular, the immunological potency may be to prevent the death of immune cells. Further, the immunological potency may be to cause suicide of immune cells after a suitable period of time has elapsed.
The new immune potency may be an immune potency in which the loss of function of immune cells is reduced.
The new immunogenic effect may be an immunogenic effect in which cytokine secretion by immune cells is regulated. In particular, the immunopotency may be promoting or inhibiting cytokine secretion.
The novel immunogenic efficacy may be modulating the antigen binding capacity of a wild-type receptor in an immune cell. In particular, the immunopotency may be to improve the specificity of the wild-type receptor for a particular antigen.
In addition, the immune cells that are functionally manipulated may be immune cells that are manipulated such that the function of the immunomodulatory factors is impaired.
Here, the function of the immunomodulatory factors can be lost or impaired by the compounds.
The compounds may bind to the immunomodulatory factors or to specific proteins that interact with the immunomodulatory factors and block the function of the immunomodulatory factors.
In addition, the compounds may bind to and artificially modify the three-dimensional structure of the immune modulatory factors, thereby preventing their normal function.
Alternatively, the function of the immune modulator may be lost or damaged by modification of a protein that interacts with the immune modulator.
As an embodiment disclosed herein, the manipulated immune cells may be functionally manipulated immune cells in which the immunomodulatory genes are manipulated manually.
Here, the immune regulatory gene may be a PD-1 gene, CTLA-4 gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene, PPP2r2d gene, TET2 gene, PSGL-1 gene, A20 gene and/or KDM6A gene.
The functionally manipulated immune cells can be manipulated by compositions for gene manipulation.
The explanation about the above-mentioned composition for gene manipulation is as described above.
A functionally manipulated immune cell may comprise one or more artificially manipulated or modified immune modulatory genes.
Here, the artificially modified immunoregulatory gene may comprise one or more of the following modifications in the target sequence or in a region of the nucleotide sequence of 1bp to 50bp adjacent to the 5 'end and/or 3' end of the target sequence:
i) a deletion of one or more nucleotides;
ii) replacing one or more nucleotides with a nucleotide different from the wild-type gene;
iii) insertion of one or more nucleotides; or
iv) a combination of two or more selected from the above i) -iii).
In one example, a functionally manipulated immune cell may comprise one or more artificially manipulated or modified immune modulatory genes.
Here, the immunoregulatory gene which is artificially manipulated or modified may comprise a deletion of one or more nucleotides in the target sequence or in a region of the nucleotide sequence of 1bp to 50bp adjacent to the 5 'end and/or 3' end of the target sequence.
For example, an immunomodulatory gene that is artificially manipulated or modified can comprise a deletion of one or more nucleotides in a region of the nucleotide sequence located in the target sequence.
Here, the deleted nucleotide may be a 1bp-50bp nucleotide in which the nucleotide is continuous, discontinuous, or a mixture of both forms (i.e., continuous and discontinuous). For example, the deleted nucleotide can be a 1bp nucleotide located in the target sequence. Alternatively, the deleted nucleotide may be a 1bp nucleotide located in the target sequence. Alternatively, the deleted nucleotide may be a contiguous 3bp nucleotide. Alternatively, the deleted nucleotide may be a discontinuous 4bp nucleotide located in the target sequence, wherein the discontinuous 4bp nucleotide may be a 1bp nucleotide and a continuous 3bp nucleotide, or a continuous 2bp nucleotide and another continuous 2bp nucleotide (fig. 1). For example, the deleted nucleotide can be a discrete 30bp nucleotide located in the target sequence, wherein the discrete 30bp nucleotide can be a continuous 25bp nucleotide, a continuous 4bp nucleotide, and a discrete 1bp nucleotide.
Alternatively, here, the deleted nucleotide may be a nucleotide fragment comprising more than 2bp of nucleotides. The nucleotide fragment can be 2bp-5bp, 6bp-10bp, 11bp-15bp, 16bp-20bp, 21bp-25bp, 26bp-30bp, 31bp-35bp, 36bp-40bp, 41bp-45bp or 46bp-50 bp. For example, the deleted nucleotide may be a 2bp nucleotide fragment located in the target sequence. Alternatively, the deleted nucleotide may be a 10bp nucleotide fragment located in the target sequence. Alternatively, the deleted nucleotide may be a 16bp nucleotide fragment located in the target sequence (FIG. 2).
Alternatively, here, the deleted nucleotide may be a nucleotide fragment comprising more than 2bp of nucleotides. Here, the nucleotide fragment containing 2bp or more nucleotides may be a single nucleotide fragment having a discontinuous nucleotide sequence (i.e., having one or more nucleotide sequence gaps), and two or more deletion regions may be generated from two or more deleted nucleotide fragments. For example, the nucleotides deleted may be a 2bp nucleotide fragment and a 6bp nucleotide fragment located in the target sequence. Alternatively, the deleted nucleotides may be a 12bp nucleotide fragment and a 6bp nucleotide fragment located in the target sequence (FIG. 3).
In another example, the immune modulatory gene that is manipulated or modified by hand may comprise a deletion of one or more nucleotides in a region of 1bp to 50bp nucleotide sequence adjacent to the 5 'end and/or 3' end of the target sequence.
Here, the deleted nucleotide may be a 1bp-50bp nucleotide in which the nucleotide is continuous, discontinuous, or a mixture of both forms (i.e., continuous and discontinuous). For example, the deleted nucleotide can be a 1bp nucleotide located in the target sequence. Alternatively, the deleted nucleotide may be a contiguous 4bp nucleotide located adjacent the 3' end of the target sequence. Alternatively, the deleted nucleotides can be discontinuous 4bp nucleotides located adjacent to the 5 'end and/or 3' end of the target sequence, wherein the discontinuous 4bp nucleotides can be continuous 3bp nucleotides located adjacent to the 5 'end of the target sequence and 1bp nucleotides located adjacent to the 3' end of the target sequence (FIG. 4). For example, the deleted nucleotide can be a discontinuous 25bp nucleotide located in the target sequence, wherein the discontinuous 25bp nucleotide can be a continuous 15bp nucleotide, a continuous 8bp nucleotide, a discontinuous 1bp nucleotide, and a discontinuous 1bp nucleotide.
Alternatively, the deleted nucleotide may be a nucleotide fragment comprising consecutive nucleotides of 2bp or more. The nucleotide fragment can be 2bp-5bp, 6bp-10bp, 11bp-15bp, 16bp-20bp, 21bp-25bp, 26bp-30bp, 31bp-35bp, 36bp-40bp, 41bp-45bp or 46bp-50 bp. For example, the deleted nucleotide can be a 2bp nucleotide fragment located adjacent to the 3' end of the target sequence. Alternatively, the deleted nucleotide may be a 10bp nucleotide fragment located adjacent to the 5' end of the target sequence. Alternatively, the deleted nucleotide may be a20 bp nucleotide fragment located adjacent to the 3' end of the target sequence (FIG. 5).
Alternatively, here, the deleted nucleotide may be a nucleotide fragment comprising more than 2bp of nucleotides. Here, the nucleotide fragment containing 2bp or more nucleotides may be a single nucleotide fragment having a discontinuous nucleotide sequence (i.e., having one or more nucleotide sequence gaps), and two or more deleted nucleotide fragments may be used to generate two or more deleted regions. For example, the deleted nucleotides can be a 3bp nucleotide fragment located adjacent to the 5 'end of the target sequence and a 6bp nucleotide fragment located adjacent to the 3' end of the target sequence. Alternatively, the deleted nucleotides may be a 12bp nucleotide fragment and a 6bp nucleotide fragment located adjacent to the 3' end of the target sequence (FIG. 6).
In yet another example, the artificially manipulated or modified immunomodulatory gene may comprise a deletion of one or more nucleotides in the target sequence and in a region of 1bp to 50bp nucleotide sequence located adjacent to the 5 'end and/or 3' end of the target sequence.
Here, the deleted nucleotide may be a 1bp-50bp nucleotide in which the nucleotide is continuous, discontinuous, or a mixture of both forms (i.e., continuous and discontinuous). For example, the deleted nucleotide can be a contiguous 4bp nucleotide located in the target sequence and adjacent to the 3' end of the target sequence. Alternatively, the deleted nucleotides can be discontinuous 3bp nucleotides located in the target sequence and in a position adjacent to the 3 'end of the target sequence, and the discontinuous 3bp nucleotides can be continuous 2bp nucleotides located in the target sequence and 1bp nucleotides located adjacent to the 3' end of the target sequence (fig. 7). For example, the deleted nucleotides can be discrete 40bp nucleotides located in the target sequence, and the discrete 25bp nucleotides can be continuous 10bp nucleotides, continuous 8bp nucleotides, and discrete 5bp (discrete 1bp, and 1bp) nucleotides.
Alternatively, here, the deleted nucleotide may be a nucleotide fragment comprising more than 2bp of nucleotides. The nucleotide fragment can be 2bp-5bp, 6bp-10bp, 11bp-15bp, 16bp-20bp, 21bp-25bp, 26bp-30bp, 31bp-35bp, 36bp-40bp, 41bp-45bp or 46bp-50 bp. For example, the deleted nucleotide can be a25 bp nucleotide fragment located in the target sequence and in a position adjacent to the 3' end of the target sequence. Alternatively, the deleted nucleotides may be 35bp nucleotide fragments located in the target sequence and in positions adjacent to the 5 'and 3' ends of the target sequence (FIG. 8).
Alternatively, here, the deleted nucleotide may be a fragment of two or more nucleotides. Here, two or more nucleotide fragments may be a single nucleotide fragment having a discontinuous nucleotide sequence (i.e., having one or more nucleotide sequence gaps), and two or more deleted nucleotide fragments may be used to generate two or more deleted regions. For example, the deleted nucleotides can be a 6bp nucleotide fragment located in the target sequence and in a position adjacent to the 5 'end of the target sequence, and a 13bp nucleotide fragment located in the target sequence and in a position adjacent to the 3' end of the target sequence. Alternatively, the deleted nucleotides may be a 17bp nucleotide fragment located in the target sequence and in a position adjacent to the 3 'end of the target sequence, and a 4bp nucleotide fragment located adjacent to the 3' end of the target sequence (FIG. 9).
In another example, a functionally manipulated immune cell may comprise one or more artificially manipulated or modified immune modulatory genes.
Here, the immune modulatory gene which is artificially manipulated or modified may comprise an insertion of one or more nucleotides in the target sequence or in a region of nucleotide sequence of 1bp to 50bp located adjacent to the 5 'end and/or 3' end of the target sequence.
For example, an immune modulatory gene that has been artificially manipulated or modified can comprise an insertion of one or more nucleotides in a region of the nucleotide sequence that is located in the target sequence.
Here, the inserted nucleotide may be a 1bp-50bp nucleotide in which the nucleotide is continuous, discontinuous, or a mixture of both forms (i.e., continuous and discontinuous). For example, the inserted nucleotide may be a contiguous 2bp nucleotide inserted in the region of the nucleotide sequence of the target sequence. Alternatively, the inserted nucleotide may be a discontinuous 3bp nucleotide inserted in a region of the nucleotide sequence in the target sequence, and the discontinuous 3bp nucleotide may be a 1bp nucleotide and a continuous 2bp nucleotide. Alternatively, the inserted nucleotide may be a discontinuous 4bp nucleotide inserted in a nucleotide sequence region in the target sequence, and the discontinuous 4bp nucleotide may be a 1bp nucleotide, a continuous 2bp nucleotide and another 1bp nucleotide (fig. 10). For example, the inserted nucleotide may be a discontinuous 30bp nucleotide inserted in a nucleotide sequence region in the target sequence, and the discontinuous 30bp nucleotide may be a continuous 15bp nucleotide, a continuous 12bp nucleotide, and a discontinuous 3bp (discontinuous 1bp, and 1bp) nucleotide.
Alternatively, the inserted nucleotide may be a nucleotide fragment comprising contiguous nucleotides of 5bp or more. The nucleotide fragment can be 5bp-10bp, 11bp-50bp, 50bp-100bp, 100bp-200bp, 200bp-300bp, 300bp-400bp, 400bp-500bp, 500bp-750bp, or 750bp-1000 bp. For example, the inserted nucleotide may be a 10bp nucleotide fragment inserted in a region of the nucleotide sequence in the target sequence. Alternatively, the inserted nucleotide may be a 28bp nucleotide fragment inserted in a region of the nucleotide sequence in the target sequence (FIG. 11).
Alternatively, the inserted nucleotide may be a partial or complete nucleotide sequence of a specific gene herein. The specific gene may be a gene introduced from an external region not contained in a subject (e.g., human cell) containing the immune regulatory gene. Alternatively, the specific gene may be a gene present in a subject (e.g., a human cell) comprising an immunomodulatory gene, e.g., a gene present in the genome of a human cell. For example, the inserted nucleotide may be a partial nucleotide sequence of an exogenous gene inserted in a nucleotide sequence region in the target sequence. Alternatively, the inserted nucleotide may be the entire nucleotide sequence of the exogenous gene inserted in the region of the nucleotide sequence in the target sequence. Alternatively, the inserted nucleotide may be a partial nucleotide sequence of an endogenous gene inserted in a nucleotide sequence region in the target sequence, the endogenous gene may be an allele of a target gene (i.e., an immunomodulatory gene), or a gene other than the target gene. Alternatively, the inserted nucleotide may be the entire nucleotide sequence of an endogenous gene inserted in a nucleotide sequence region in the target sequence, and the endogenous gene may be an allele of a target gene (i.e., an immunomodulatory gene) or a gene other than the target gene (fig. 12).
In another example, the artificially manipulated or modified immunomodulatory gene may comprise an insertion of one or more nucleotides in the target sequence or in a region of 1bp to 50bp nucleotide sequence located adjacent to the 5 'end and/or 3' end of the target sequence.
Here, the inserted nucleotide may be a 1bp-50bp nucleotide in which the nucleotide is continuous, discontinuous, or a mixture of both forms (i.e., continuous and discontinuous). For example, the inserted nucleotide may be a contiguous 2bp nucleotide inserted in the region of the nucleotide sequence located adjacent to the 5' end of the target sequence. Alternatively, the inserted nucleotide may be a discontinuous 3bp nucleotide inserted in a region of the nucleotide sequence located adjacent to the 3' end of the target sequence, and the discontinuous 3bp nucleotide may be a 1bp nucleotide and a continuous 2bp nucleotide (fig. 13). For example, the inserted nucleotide may be a discontinuous 40bp nucleotide inserted in a nucleotide region of the target sequence, and the discontinuous 40bp nucleotide may be a continuous 15bp nucleotide, a continuous 20bp nucleotide and a continuous 5bp nucleotide.
Alternatively, the inserted nucleotide may be a nucleotide fragment comprising contiguous nucleotides of 5bp or more. The nucleotide fragment can be 5bp-10bp, 11bp-50bp, 50bp-100bp, 100bp-200bp, 200bp-300bp, 300bp-400bp, 400bp-500bp, 500bp-750bp or 750bp-1000 bp. For example, the inserted nucleotide may be a 22bp nucleotide fragment inserted in the region of the nucleotide sequence located near the 5' end of the target sequence. Alternatively, the inserted nucleotide may be a37 bp nucleotide fragment inserted in the region of the nucleotide sequence located near the 3' end of the target sequence (FIG. 14).
Alternatively, the inserted nucleotide may be a partial or complete nucleotide sequence of a specific gene herein. The specific gene may be a gene introduced from an external region not contained in a subject (e.g., human cell) containing the immune regulatory gene. Alternatively, the specific gene may be a gene contained in a subject (e.g., a human cell) comprising an immunomodulatory gene, such as a gene present in the genome of a human cell. For example, the inserted nucleotide may be a partial nucleotide sequence of an exogenous gene inserted in a region of the nucleotide sequence located adjacent to the 5' end of the target sequence. Alternatively, the inserted nucleotide may be the entire nucleotide sequence of the exogenous gene inserted in the region of the nucleotide sequence located near the 3' end of the target sequence. Alternatively, the inserted nucleotide may be a partial nucleotide sequence of an endogenous gene inserted in a region of a nucleotide sequence located near the 5' end of the target sequence, and the endogenous gene may be an allele of a target gene (i.e., an immunomodulatory gene), or a gene other than the target gene. Alternatively, the inserted nucleotide may be the entire nucleotide sequence in the endogenous gene inserted in the nucleotide sequence region located near the 3' end of the target sequence, and the endogenous gene may be an allele of the target gene (i.e., an immunomodulatory gene) or a gene other than the target gene (fig. 15).
In yet another example, a functionally manipulated immune cell can comprise one or more artificially manipulated or modified immune modulatory genes.
Here, the immunoregulatory gene which is artificially manipulated or modified may comprise a deletion and insertion of one or more nucleotides in the target sequence or in a region of 1bp to 50bp nucleotide sequence located adjacent to the 5 'end and/or 3' end of the target sequence.
For example, an immune modulatory gene that has been artificially manipulated or modified can comprise deletions and insertions of one or more nucleotides in the region of the nucleotide sequence located in the target sequence.
Here, the deleted nucleotide may be a 1bp-50bp nucleotide in which the nucleotide is continuous, discontinuous, or a mixture of both forms (i.e., continuous and discontinuous).
Here, the inserted nucleotide may be a 1bp-50bp nucleotide in which the nucleotide is continuous, discontinuous, or a mixture of both forms (i.e., continuous and discontinuous); a nucleotide fragment; or a part or all of the nucleotide sequence of a specific gene, and the deletion and insertion may be performed sequentially or simultaneously.
The inserted nucleotide fragment can be 5bp-10bp, 11bp-50bp, 50bp-100bp, 100bp-200bp, 200bp-300bp, 300bp-400bp, 400bp-500bp, 500bp-750bp or 750bp-1000 bp.
The specific gene may be a gene introduced from an external region not contained in a subject (e.g., human cell) containing the immune regulatory gene. Alternatively, the specific gene may be a gene contained in a subject (e.g., a human cell) comprising an immunomodulatory gene, such as a gene present in the genome of a human cell.
For example, deletions and insertions of nucleotides may occur at similar positions in the target sequence, and the deleted nucleotide may be a 1bp nucleotide located in the target sequence; in this case, the inserted nucleotide may be a continuous 2bp nucleotide inserted in the position of the deleted nucleotide sequence. Alternatively, the deleted nucleotide may be a contiguous 3bp nucleotide located in the target sequence; in this case, the inserted nucleotide may be a contiguous 20bp nucleotide fragment inserted in the position of the deleted nucleotide sequence. Alternatively, the deleted nucleotide may be a contiguous 2bp nucleotide located in the target sequence; in this case, the inserted nucleotide may be a partial nucleotide sequence of the exogenous gene inserted in the position of the deleted nucleotide sequence. Alternatively, the deleted nucleotide may be a contiguous 3bp nucleotide located in the target sequence; in this case, the inserted nucleotide may be the entire nucleotide sequence of an endogenous gene inserted at the position of the deleted nucleotide sequence, and the endogenous gene may be an allele of a target gene (i.e., an immunomodulatory gene) or a gene other than the target gene (fig. 16).
For example, deletions and insertions of nucleotides may occur at different positions in the target sequence, and the deleted nucleotides may be consecutive 4bp nucleotides located in the target sequence, in which case the inserted nucleotides may be consecutive 12bp nucleotide fragments inserted in different positions in the target sequence that are not deleted. Alternatively, the deleted nucleotide may be a contiguous 5bp nucleotide located in the target sequence; in this case, the inserted nucleotide may be a partial nucleotide sequence of an endogenous gene inserted in a different position not deleted in the target sequence, and the endogenous gene may be an allele of a target gene (i.e., an immunomodulatory gene) or a gene other than the target gene (fig. 17).
For example, deletions and insertions of nucleotides may occur at similar or different positions in the target sequence, and the deleted nucleotides may be 1bp and consecutive 4bp nucleotides located in the target sequence; in this case, the inserted nucleotide may be a continuous 10bp nucleotide fragment inserted in one of two deletion positions (i.e., the position where the 1bp nucleotide is deleted) of the target sequence. Alternatively, the deleted nucleotides may be consecutive 5bp and 1bp nucleotides located in the target sequence; in this case, the inserted nucleotide may be the entire nucleotide sequence of an endogenous gene inserted in one of the two deletion positions (i.e., the position of consecutive 5bp nucleotide deletions), which may be an allele of a target gene (i.e., an immunomodulatory gene), or a gene other than the target gene (fig. 18).
Alternatively, here, the deleted nucleotide may be a nucleotide fragment comprising more than 2bp of nucleotides.
The deleted nucleotide fragment can be 2bp-5bp, 6bp-10bp, 11bp-15bp, 16bp-20bp, 21bp-25bp, 26bp-30bp, 31bp-35bp, 36bp-40bp, 41bp-45bp or 46bp-50 bp.
Here, the inserted nucleotide may be a 1bp-50bp nucleotide in which the nucleotide is continuous, discontinuous, or a mixture of both forms (i.e., continuous and discontinuous); a nucleotide fragment; or a part or the whole of the nucleotide sequence of a specific gene, and the deletion and the insertion may be performed sequentially or simultaneously.
For example, deletions and insertions of nucleotides may occur at similar positions in the target sequence, and the deleted nucleotides may be a 10bp nucleotide fragment located in the target sequence; in this case, the inserted nucleotide may be a continuous 2bp nucleotide inserted in the position of the deleted nucleotide sequence. Alternatively, the deleted nucleotide may be a contiguous 17bp nucleotide fragment located in the target sequence; in this case, the inserted nucleotide may be a contiguous 20bp nucleotide fragment inserted in the position of the deleted nucleotide sequence. Alternatively, the deleted nucleotide may be a 15bp nucleotide fragment located in the target sequence; in this case, the inserted nucleotide may be a partial nucleotide sequence of an endogenous gene inserted in the position of the deleted nucleotide sequence, and the endogenous gene may be an allele of a target gene (i.e., an immunomodulatory gene) or a gene other than the target gene. Alternatively, the deleted nucleotide may be a 7bp nucleotide fragment located in the target sequence; in this case, the inserted nucleotide may be the entire nucleotide sequence of the exogenous gene inserted at the position of the deleted nucleotide sequence (fig. 19).
Alternatively, here, the deleted nucleotide may be a nucleotide fragment comprising more than 2bp of nucleotides.
Here, the inserted nucleotide may be a 1bp-50bp nucleotide in which the nucleotide is continuous, discontinuous, or a mixture of both forms (i.e., continuous and discontinuous); a nucleotide fragment; or a part or the whole of the nucleotide sequence of a specific gene, and the deletion and the insertion may be performed sequentially or simultaneously. Furthermore, the insertion may occur in part or all of two or more deletion regions.
For example, deletions and insertions of nucleotides may occur at similar and/or different positions in the target sequence, and the deleted nucleotides may be a 6bp nucleotide fragment and a 12bp nucleotide fragment located in the target sequence; in this case, the inserted nucleotide may be a 15bp nucleotide fragment in one of the two deletion positions of the target sequence (i.e., the position where the 6bp nucleotide is deleted). Alternatively, the deleted nucleotides may be a 12bp nucleotide fragment and an 8bp nucleotide fragment located in the target sequence; in this case, the inserted nucleotides may be 13bp nucleotide fragments inserted in the two deleted nucleotide sequences, respectively, i.e., a 13bp nucleotide fragment inserted in the position of the deleted 12bp nucleotide fragment, and a 13bp nucleotide fragment inserted in the position of the deleted 8bp nucleotide. Alternatively, the deleted nucleotides may be a 7bp nucleotide fragment and an 8bp nucleotide fragment located in the target sequence; in this case, the inserted nucleotides may be a partial or complete nucleotide sequence of the endogenous gene inserted in the two deleted nucleotide sequences, respectively, i.e., the complete nucleotide sequence of the endogenous gene inserted in the position of the deleted 7bp nucleotide fragment and the partial nucleotide sequence of the endogenous gene inserted in the position of the deleted 8bp nucleotide fragment. Alternatively, the deleted nucleotides may be a 9bp nucleotide fragment and an 8bp nucleotide fragment located in the target sequence; in this case, the inserted nucleotides may be an 8bp nucleotide fragment inserted in the two deleted nucleotide sequences and the entire or partial nucleotide sequence of the foreign gene, respectively, i.e., an 8bp nucleotide fragment inserted in the position of the deleted 9bp nucleotide fragment and a partial nucleotide sequence of the foreign gene inserted in the position of the deleted 8bp nucleotide fragment (fig. 20).
In another example, the immune modulatory gene that is artificially manipulated or modified can comprise deletions and insertions of one or more nucleotides in the region of a 1bp-50bp nucleotide sequence located adjacent to the 5 'end and/or 3' end of the target sequence.
Here, the deleted nucleotide may be a 1bp-50bp nucleotide in which the nucleotide is continuous, discontinuous, or a mixture of both forms (i.e., continuous and discontinuous).
Here, the inserted nucleotide may be a 1bp-50bp nucleotide in which the nucleotide is continuous, discontinuous, or a mixture of both forms (i.e., continuous and discontinuous); a nucleotide fragment; or a part or the whole of the nucleotide sequence of a specific gene, and the deletion and the insertion may be performed sequentially or simultaneously.
The inserted nucleotide fragment can be 5bp-10bp, 11bp-50bp, 50bp-100bp, 100bp-200bp, 200bp-300bp, 300bp-400bp, 400bp-500bp, 500bp-750bp or 750bp-1000 bp.
The specific gene may be a gene introduced from an external region not contained in a subject (e.g., human cell) containing the immune regulatory gene. Alternatively, the specific gene may be a gene contained in a subject (e.g., a human cell) comprising an immunomodulatory gene, such as a gene present in the genome of a human cell.
For example, deletions and insertions of nucleotides can occur at similar positions adjacent the 5' and/or 3' end of the target sequence, and the deleted nucleotide can be a 1bp nucleotide located adjacent the 3' end of the target sequence; in this case, the inserted nucleotide may be a continuous 2bp nucleotide inserted in the position of the deleted nucleotide sequence. Alternatively, the deleted nucleotide may be a contiguous 3bp nucleotide located adjacent to the 5' end of the target sequence; in this case, the inserted nucleotide may be a20 bp nucleotide inserted in the position of the deleted nucleotide sequence. Alternatively, the deleted nucleotide may be a contiguous 3bp nucleotide located adjacent the 3' end of the target sequence; in this case, the inserted nucleotide may be a partial nucleotide sequence of the endogenous gene inserted in the position of the deleted nucleotide sequence. Alternatively, the deleted nucleotide may be a contiguous 2bp nucleotide located adjacent to the 5' end of the target sequence; in this case, the inserted nucleotide may be the entire nucleotide sequence of the exogenous gene inserted at the position of the deleted nucleotide sequence. Alternatively, the deleted nucleotides may be 1bp and consecutive 4bp nucleotides located adjacent to the 3' end of the target sequence; in this case, the inserted nucleotides may be the entire nucleotide sequence of the endogenous gene and the consecutive 4bp nucleotide sequences inserted in the two deleted nucleotide sequences, respectively, i.e., the entire nucleotide sequence of the endogenous gene inserted in the position of the deleted 1bp nucleotide sequence and the consecutive 4bp nucleotide sequences inserted in the position of the deleted consecutive 4bp nucleotides (fig. 21).
For example, the deletion and insertion of nucleotides may occur at similar or different positions in the nucleotide sequence located adjacent to the 5 'end and/or 3' end of the target sequence, and the deleted nucleotides may be 1bp of nucleotides located adjacent to the 5 'end of the target sequence and consecutive 3bp of nucleotides located adjacent to the 3' end of the target sequence; in this case, the inserted nucleotide may be an 8bp nucleotide fragment inserted in one of the positions of the deleted nucleotide sequence (i.e., the positions of the deleted consecutive 3bp nucleotides). Alternatively, the deleted nucleotide may be a contiguous 4bp nucleotide located adjacent to the 5' end of the target sequence; in this case, the inserted nucleotide may be a partial nucleotide sequence of the endogenous gene inserted in a different position adjacent to the 3' end of the target sequence that is not deleted (fig. 22).
Alternatively, here, the deleted nucleotide may be a nucleotide fragment comprising more than 2bp of nucleotides.
The deleted nucleotide fragment can be 2bp-5bp, 6bp-10bp, 11bp-15bp, 16bp-20bp, 21bp-25bp, 26bp-30bp, 31bp-35bp, 36bp-40bp, 41bp-45bp or 46bp-50 bp.
Here, the inserted nucleotide may be a 1bp-50bp nucleotide in which the nucleotide is continuous, discontinuous, or a mixture of both forms (i.e., continuous and discontinuous); a nucleotide fragment; or a part or the whole of the nucleotide sequence of a specific gene, and the deletion and the insertion may be performed sequentially or simultaneously.
For example, the deletion and insertion of nucleotides can occur at similar positions adjacent to the 5' and/or 3' end of the target sequence, and the deleted nucleotides can be a 17bp nucleotide fragment located adjacent to the 3' end of the target sequence; in this case, the inserted nucleotide may be a continuous 2bp nucleotide inserted in the position of the deleted nucleotide sequence. Alternatively, the deleted nucleotide may be a 15bp nucleotide fragment located adjacent to the 5' end of the target sequence; in this case, the inserted nucleotide may be a 30bp nucleotide fragment inserted in the position of the deleted nucleotide sequence. Alternatively, the deleted nucleotide may be a 15bp nucleotide fragment located adjacent to the 5' end of the target sequence; in this case, the inserted nucleotide may be a partial nucleotide sequence of the endogenous gene inserted in the position of the deleted nucleotide sequence. Alternatively, the deleted nucleotide may be a25 bp nucleotide fragment located adjacent to the 3' end of the target sequence; in this case, the inserted nucleotide may be the entire nucleotide sequence of the endogenous gene inserted in the position of the deleted nucleotide sequence (fig. 23).
Alternatively, here, the deleted nucleotide may be a nucleotide fragment comprising more than 2bp of nucleotides.
Here, the inserted nucleotide may be 1bp, 2bp, 3bp, 4bp or 5 bp; a nucleotide fragment; or a part or the whole of the nucleotide sequence of a specific gene, and the deletion and the insertion may be performed sequentially or simultaneously. Alternatively, the insertion may occur in part or all of two or more deletion areas.
For example, deletions and insertions of nucleotides can occur at similar positions adjacent the 5 'and/or 3' end of the target sequence, and the deleted nucleotides can be a 7bp nucleotide fragment located adjacent the 5 'end of the target sequence and an 18bp nucleotide fragment located adjacent the 3' end of the target sequence; in this case, the inserted nucleotides may be a partial nucleotide sequence of the exogenous gene and a 12bp nucleotide fragment inserted in the two deleted nucleotide sequences, respectively, that is, a partial nucleotide sequence of the exogenous gene inserted in the position of the deleted 7bp nucleotide fragment and a 12bp nucleotide fragment inserted in the position of the deleted 18bp nucleotide fragment. Alternatively, the deleted nucleotides may be a 10bp nucleotide fragment located adjacent the 3 'end of the target sequence and a 6bp nucleotide fragment located adjacent the 5' end of the target sequence; in this case, the inserted nucleotides may be the entire nucleotide sequence of the endogenous gene and consecutive 4bp nucleotides inserted in the two deleted nucleotide sequences, respectively, i.e., the entire nucleotide sequence of the endogenous gene inserted in the position of the deleted 10bp nucleotide fragment and the consecutive 4bp nucleotides inserted in the position of the deleted 6bp nucleotide fragment (fig. 24).
In yet another example, the immune modulatory gene that is manipulated or modified by man may comprise deletions and insertions of one or more nucleotides in the target sequence and in the region of the 1bp-50bp nucleotide sequence located adjacent to the 5 'end and/or 3' end of the target sequence.
Here, the deleted nucleotide may be a 1bp-50bp nucleotide in which the nucleotide is continuous, discontinuous, or a mixture of both forms (i.e., continuous and discontinuous).
Here, the inserted nucleotide may be a 1bp-50bp nucleotide in which the nucleotide is continuous, discontinuous, or a mixture of both forms (i.e., continuous and discontinuous); a nucleotide fragment; or a part or the whole of the nucleotide sequence of a specific gene, and the deletion and the insertion may be performed sequentially or simultaneously.
The inserted nucleotide fragment can be 5bp-10bp, 11bp-50bp, 50bp-100bp, 100bp-200bp, 200bp-300bp, 300bp-400bp, 400bp-500bp, 500bp-750bp or 750bp-1000 bp.
The specific gene may be a gene introduced from an external region not contained in a subject (e.g., human cell) containing the immune regulatory gene. Alternatively, the specific gene may be a gene contained in a subject (e.g., a human cell) comprising an immunomodulatory gene, such as a gene present in the genome of a human cell.
For example, deletions and insertions of nucleotides may occur at similar positions in the nucleotide sequence located in the target sequence and adjacent to the 5' end and/or 3' end of the target sequence, and the deleted nucleotides may be consecutive 4bp nucleotides located in the target sequence and adjacent to the 3' end of the target sequence; in this case, the inserted nucleotide may be a 5bp nucleotide fragment inserted in the position of the deleted nucleotide sequence. Alternatively, the deleted nucleotides can be consecutive 2bp nucleotides located in the target sequence and consecutive 2bp nucleotides located adjacent to the 3' end of the target sequence; in this case, the inserted nucleotides may be a 10bp nucleotide fragment inserted in the two deleted nucleotide sequences and a partial nucleotide sequence of the exogenous gene, respectively, i.e., a 10bp nucleotide fragment inserted in the position of the deleted continuous 2bp nucleotide sequence located in the target sequence, and a partial nucleotide sequence of the exogenous gene inserted in the position of the deleted continuous 2bp nucleotide sequence located near the 3' end of the target sequence (fig. 25).
Alternatively, here, the deleted nucleotide may be a nucleotide fragment comprising more than 2bp of nucleotides.
The deleted nucleotide fragment can be 2bp-5bp, 6bp-10bp, 11bp-15bp, 16bp-20bp, 21bp-25bp, 26bp-30bp, 31bp-35bp, 36bp-40bp, 41bp-45bp or 46bp-50 bp.
Here, the inserted nucleotide may be a 1bp-50bp nucleotide in which the nucleotide is continuous, discontinuous, or a mixture of both forms (i.e., continuous and discontinuous); a nucleotide fragment; or a part or the whole of the nucleotide sequence of a specific gene, and the deletion and the insertion may be performed sequentially or simultaneously.
For example, deletions and insertions of nucleotides may occur at similar positions in the nucleotide sequence located in the target sequence and adjacent to the 5' end and/or 3' end of the target sequence, and the deleted nucleotides may be contiguous 25bp nucleotides located in the target sequence and adjacent to the 3' end of the target sequence; in this case, the inserted nucleotide may be the entire nucleotide sequence of the exogenous gene inserted at the position of the deleted nucleotide sequence. Alternatively, the deleted nucleotide may be a 30bp nucleotide fragment located in and adjacent to the 5 'or 3' end of the target sequence; in this case, the inserted nucleotide may be a 45bp nucleotide fragment inserted at the position of the deleted nucleotide sequence. (FIG. 26).
Alternatively, here, the deleted nucleotide may be a nucleotide fragment comprising more than 2bp of nucleotides.
Here, the inserted nucleotide may be a 1bp-50bp nucleotide in which the nucleotide is continuous, discontinuous, or a mixture of both forms (i.e., continuous and discontinuous); a nucleotide fragment; or a part or the whole of the nucleotide sequence of a specific gene, and the deletion and the insertion may be performed sequentially or simultaneously. Furthermore, the insertion may occur in part or all of two or more deletion regions.
For example, deletions and insertions of nucleotides may occur at similar positions in the nucleotide sequence located within the target sequence and adjacent to the 5 'end and/or 3' end of the target sequence, and the deleted nucleotides may be 25bp nucleotide fragments located within the target sequence and adjacent to the 3 'end of the target sequence, 6bp nucleotide fragments located adjacent to the 3' end of the target sequence; in this case, the inserted nucleotides may be the entire nucleotide sequence of the endogenous gene and a20 bp nucleotide fragment inserted in the two deleted nucleotide sequences, respectively, i.e., the entire nucleotide sequence of the endogenous gene inserted in the position of the deleted 25bp nucleotide fragment and the 20bp nucleotide fragment inserted in the position of the deleted 6bp nucleotide fragment. Alternatively, the deleted nucleotides can be a 10bp nucleotide fragment located in and adjacent to the 5 'end of the target sequence and a 22bp nucleotide fragment located in and adjacent to the 3' end of the target sequence; in this case, the inserted nucleotides may be a partial nucleotide sequence of the endogenous gene and a full nucleotide sequence of the exogenous gene inserted in the two deleted nucleotide sequences, respectively, i.e., a partial nucleotide sequence of the endogenous gene inserted in the position of the deleted 10bp nucleotide fragment and a full nucleotide sequence of the exogenous gene inserted in the position of the deleted 22bp nucleotide fragment (fig. 27).
A functionally manipulated immune cell may comprise one or more artificially manipulated or modified immune modulatory genes.
The artificially manipulated or modified immunomodulatory gene may comprise one or more of the following modifications in a contiguous 1bp to 50bp nucleotide sequence region located at the 5 'end and/or 3' end of a PAM sequence present in the nucleotide sequence adjacent to the immunomodulatory gene:
i) a deletion of one or more nucleotides;
ii) replacing one or more nucleotides with a nucleotide different from the wild-type gene;
iii) insertion of one or more nucleotides; or
iv) a combination of two or more selected from the above i) -iii).
In one example, when the CRISPR enzyme is SpCas9 protein, the artificially manipulated or modified immunomodulatory gene may comprise one or more of the following modifications in a contiguous 1bp-50bp, 1bp-40bp, 1bp-30bp, or 1bp-25bp nucleotide sequence region located at the 5 'end and/or 3' end of a 5'-NGG-3' (N is A, T, G or C) PAM sequence present in the nucleotide sequence adjacent to the immunomodulatory gene:
i) a deletion of one or more nucleotides;
ii) replacing one or more nucleotides with a nucleotide different from the wild-type gene;
iii) insertion of one or more nucleotides; or
iv) a combination of two or more selected from the above i) -iii).
In another example, when the CRISPR enzyme is a CjCas9 protein, the artificially manipulated or modified immunomodulatory gene may comprise one or more of the following modifications in a contiguous 1bp-50bp, 1bp-40bp, 1bp-30bp, or 1bp-25bp nucleotide sequence region located at the 5 'end and/or 3' end of a 5'-NNNNRYAC-3' (N is each independently A, T, C or G; R is a or G; Y is C or T) PAM sequence present in a nucleotide sequence adjacent to the immunomodulatory gene:
i) a deletion of one or more nucleotides;
ii) replacing one or more nucleotides with a nucleotide different from the wild-type gene;
iii) insertion of one or more nucleotides; or
iv) a combination of two or more selected from the above i) -iii).
In yet another example, when the CRISPR enzyme is a StCas9 protein, the artificially manipulated or modified immunomodulatory gene may comprise one or more of the following modifications in a contiguous 1bp-50bp, 1bp-40bp, 1bp-30bp, or 1bp-25bp nucleotide sequence region located at the 5' -NNAGaaw-3' (N is each independently A, T, C or G; W is a or T) and/or 3' end of the PAM sequence present in the nucleotide sequence adjacent to the immunomodulatory gene:
i) a deletion of one or more nucleotides;
ii) replacing one or more nucleotides with a nucleotide different from the wild-type gene;
iii) insertion of one or more nucleotides; or
iv) a combination of two or more selected from the above i) -iii).
In one example, when the CRISPR enzyme is NmCas9 protein, the artificially manipulated or modified immunomodulatory gene may comprise one or more of the following modifications in a contiguous 1bp-50bp, 1bp-40bp, 1bp-30bp, or 1bp-25bp nucleotide sequence region located at the 5 'end and/or 3' end of a 5'-NNNNGATT-3' (N is each independently A, T, C or G) PAM sequence present in a nucleotide sequence adjacent to the immunomodulatory gene:
i) a deletion of one or more nucleotides;
ii) replacing one or more nucleotides with a nucleotide different from the wild-type gene;
iii) insertion of one or more nucleotides; or
iv) a combination of two or more selected from the above i) -iii).
In another example, when the CRISPR enzyme is a SacAS9 protein, the artificially manipulated or modified immunomodulatory gene may comprise one or more of the following modifications in the contiguous 1bp-50bp, 1bp-40bp, 1bp-30bp, and 1bp-25bp nucleotide sequence regions located at the 5' -NNGRR (T) -3' (N is each independently A, T, G or C; R is A or G; and (T) is any sequence that may optionally be comprised) end and/or 3' end of the PAM sequence, present in the nucleotide sequence adjacent to the immunomodulatory gene:
i) a deletion of one or more nucleotides;
ii) replacing one or more nucleotides with a nucleotide different from the wild-type gene;
iii) insertion of one or more nucleotides; or
iv) a combination of two or more selected from the above i) -iii).
In yet another example, when the CRISPR enzyme is a Cpf1 protein, the artificially manipulated or modified immunomodulatory gene may comprise one or more of the following modifications in contiguous 1bp-50bp, 1bp-40bp, 1bp-30bp, and 1bp-25bp nucleotide sequence regions located at the 5 'end and/or 3' end of a 5'-TTN-3' (N is A, T, C or G) PAM sequence present in the nucleotide sequence adjacent to the immunomodulatory gene:
i) a deletion of one or more nucleotides;
ii) replacing one or more nucleotides with a nucleotide different from the wild-type gene;
iii) insertion of one or more nucleotides; or
iv) a combination of two or more selected from the above i) -iii).
A functionally manipulated immune cell may comprise one or more knockout artificially manipulated or modified immune modulatory genes.
Here, the knock-out may be an effect caused by artificial manipulation or modification of an immunomodulatory gene.
Here, the knockout may be inhibition of expression of a protein encoded by an immunomodulatory gene by artificial manipulation or modification.
A functionally manipulated immune cell may comprise one or more immune modulatory genes that have been artificially manipulated or modified by knockdown.
Here, the knockdown may be an effect caused by manual manipulation or modification of an immunomodulatory gene.
Here, the knockdown may be inhibition of expression of a protein encoded by an immunomodulatory gene by artificial manipulation or modification.
Functionally manipulated immune cells may comprise one or more knockin foreign nucleic acids or foreign genes.
Here, one or more knock-in foreign nucleic acids or foreign genes can be introduced by manual manipulation or modification of the immunomodulatory genes.
Here, one or more knock-in foreign nucleic acids or foreign genes may express a foreign peptide or foreign protein encoding.
In addition, the functionally manipulated immune cells can be immune cells having suppressed or suppressed expression of an immunomodulatory factor.
Here, the immunomodulatory factor may be a polypeptide or protein expressed by an immunomodulatory gene (i.e., PD-1 gene, CTLA-4 gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene, PPP2r2d gene, TET2 gene, PSGL-1 gene, A20 gene, and/or KDM6A gene).
Here, the amount of the modified immune modulator expressed by the functionally modified immune cell is reduced by at least 30% when compared to the amount of the immune modulator expressed by a wild-type immune cell (i.e., a naturally occurring immune cell). Here, the amount of the immunomodulatory factor expressed by the wild-type immune cells as a comparative standard may be an average amount of the immunomodulatory factor expressed by naturally occurring immune cells collected from healthy persons without immunological diseases, such as cancer and Acquired Immune Deficiency Syndrome (AIDS), in which case the population (i.e., the number of healthy persons from which the wild-type immune cells are obtainable) may be at least 50.
Here, the amount of the modified immune modulator expressed by the functionally modified immune cell is reduced by at least 30% when compared to the amount of the immune modulator expressed by a wild-type immune cell (i.e., an immune cell prior to manual manipulation). Here, the amount of the immunomodulatory factor expressed by the wild-type immune cell as a comparative standard may be the average amount of immunomodulatory factors expressed by wild-type immune cells (i.e., immune cells prior to manual manipulation, e.g., immune cells isolated from a human prior to treatment with a composition for immune cell manipulation or an immunomodulatory gene-targeting composition for gene manipulation).
As embodiments disclosed herein, the manipulated immune cells can be hybrid manipulated immune cells.
The term "hybrid-manipulated immune cell" is an immune cell, and refers to a functionally-manipulated immune cell in which there is one or more artificial structures that are supplemented by artificial manipulation, or a supplemented artificial structures in which artificial manipulation is performed to modify the expression of natural immune modulators or impair the function of immune modulators.
The term "immune cells that supplement an artificial structure" refers to immune cells that have one or more artificial structures supplemented.
For example, the artificial structure may be an artificial receptor.
The term "artificial receptor" refers to an artificially prepared functional entity that is not a wild-type receptor, which has the ability of antigen recognition and to perform a specific function.
Such artificial receptors may contribute to the enhancement of an immune response with improved specific antigen recognition capabilities or by generating enhanced immune response signals.
As an example, the artificial receptor may have the following composition:
(i) antigen recognition moieties
The artificial receptor comprises an antigen recognition moiety.
The term "antigen recognition moiety" is a part of an artificial receptor and refers to a region that recognizes an antigen.
The antigen recognition moiety may be one that has improved recognition of a particular antigen as compared to the wild-type receptor. In particular, the specific antigen may be an antigen of a cancer cell. In addition, the specific antigen may be an antigen of a normal cell in the body.
The antigen recognition moiety may have antigen binding affinity.
The antigen recognition moiety may generate a signal while binding the antigen. The signal may be an electrical signal. The signal may be a chemical signal.
The antigen recognition portion may comprise a signal sequence.
Signal sequence refers to a peptide sequence that allows the delivery of a protein to a specific site during protein synthesis.
The signal sequence may be located near the N-terminus of the antigen recognition portion. In particular, it may be about 100 amino acids from the N-terminus. The signal sequence may be located near the C-terminus of the antigen recognition portion. In particular, it may be about 100 amino acids from the C-terminus.
The antigen recognition moiety may have an organic functional relationship with the first signal generating moiety.
The antigen recognition portion may have homology to the antigen binding fragment (Fab) domain of the antibody.
The antigen recognition moiety may be a single chain variable fragment (scFv).
The antigen recognition portion can recognize an antigen by its own antigen, or by forming an antigen recognition structure.
The antigen recognition structure can recognize an antigen by establishing a specific structure, and one of ordinary skill in the art can easily understand the combination of monomer units (monomeric units) and monomer units constituting the specific structure. Furthermore, the antigen recognition structure may be composed of one or more than two monomeric units.
The antigen recognition structure may be a structure in which the monomer units are connected in series, or may be a structure in which the monomer units are connected in parallel.
The structure connected in series refers to a structure in which two or more monomer units are continuously connected in one direction, and the structure connected in parallel refers to a structure in which two or more monomer units are each connected at the end of one monomer unit at the same time, for example, in different directions.
For example, the monomer units may be inorganic.
The monomeric units may be biochemical ligands.
The monomeric units may have homology to the antigen recognition portion of the wild-type receptor.
The monomeric units may have homology to the antibody protein.
The monomeric unit may be an immunoglobulin heavy chain or may have homology thereto.
The monomeric unit may be an immunoglobulin light chain or may have homology thereto.
The monomeric unit may comprise a signal sequence.
Meanwhile, the monomer units may be linked by chemical bonds, or may be bound by a specific combination part.
The term "antigen recognition unit combining part" is a region where antigen recognition units are linked to each other, and when an antigen recognition structure composed of two or more antigen recognition units is present, the antigen recognition unit combining part may be present in an optional composition.
The antigen recognition unit combination portion may be a peptide. In particular, the combined fraction may have a homoserine to threonine ratio.
The antigen recognition unit combination part may be a chemical binding.
The antigen recognition unit combination portion may assist the expression of the three-dimensional structure of the antigen recognition unit by having a specific length.
The antigen recognition unit combination portion can assist the function of the antigen recognition structure by having a specific positional relationship between the antigen recognition units.
(ii) Receptor body (receptor body)
The artificial receptor comprises a receptor body.
The term "acceptor body" is a region that mediates a linkage between an antigen recognition moiety and a signaling moiety, which may be physically linked.
The receptor body may function to deliver a signal generated in the antigen recognition moiety or the signal generating moiety.
According to circumstances, the structure of the receptor body may simultaneously function as a signal generating portion.
The receptor body may function to allow the artificial receptor to be immobilized on an immune cell.
The acceptor body may comprise an amino acid helix.
The structure of the receptor body may comprise a portion having homology to a portion of a normal receptor protein present in vivo. Homology may be in the range of 50% to 100%.
The structure of the receptor body may comprise portions with homology to proteins on immune cells. Homology may be in the range of 50% to 100%.
For example, the receptor body may be the CD8 transmembrane domain.
The receptor body may be the CD28 transmembrane domain. In particular, when the second signaling moiety is CD28, CD28 may perform the functions of the second signaling moiety and the recipient host.
(iii) Signal generating section
The artificial receptor may comprise a signaling moiety.
The term "first signal generating moiety" is a moiety of an artificial receptor and refers to a moiety that generates an immune response signal.
The term "second signaling moiety" is a moiety of an artificial receptor and refers to a moiety that generates an immune response signal by interacting with the first signaling moiety or independently.
The artificial receptor may comprise a first signal generating moiety and/or a second signal generating moiety.
The artificial receptor may comprise more than two first signal generating moieties and/or second signal generating moieties, respectively.
The first signaling moiety and/or the second signaling moiety may comprise a specific sequence motif.
The sequence motif may have homology to a motif of a designated Cluster (CD) protein.
In particular, the CD protein may be CD3, CD247, and CD 79.
The sequence motif can be the amino acid sequence YxxL/I.
The sequence motif can be plural within the first signaling moiety and/or the second signaling moiety.
In particular, the first sequence motif can be located 1-200 amino acids from the start of the first signal generating portion. The second sequence motif can be located 1-200 amino acids from the start of the second signal generating portion.
Furthermore, the distance between each sequence motif can be 1-15 amino acids.
In particular, the preferred distance between each sequence motif is 6-8 amino acids.
For example, the first signal generating portion and/or the second signal generating portion may be a CD3 ζ.
The first signal generating portion and/or the second signal generating portion may be fcsry.
The first signal generating portion and/or the second signal generating portion may be a signal generating portion that generates an immune response only when a specific condition is satisfied.
The specific condition may be that the antigen recognizing portion recognizes an antigen.
The specific condition may be that the antigen recognition moiety forms a bond with the antigen.
The specific condition may be that a signal generated when the antigen recognition moiety forms a binding with the antigen is delivered.
The specific condition may be that the antigen recognizing portion recognizes the antigen or that the antigen recognizing portion is separated from the antigen in the case of binding to the antigen.
The immune response signal may be a signal associated with the growth and differentiation of immune cells.
The immune response signal may be a signal associated with death of an immune cell.
The immune response signal may be a signal that is associated with the activity of an immune cell.
The immune response signal may be a signal associated with the help of an immune cell.
The immune response signal may be specifically activated by the signal generated by the antigen recognition moiety.
The immune response signal may be a signal that modulates the expression of a gene of interest.
The immune response signal may be a signal that suppresses the immune response.
In an embodiment, the signal generation section may include an additional signal generation section.
The term "additional signaling moiety" is a portion of an artificial receptor and refers to a region that produces an additional immune response signal relative to the immune response signal produced by the first signaling moiety and/or the second signaling moiety.
Hereinafter, the extra-signal generation section is referred to as an nth signal generation section (n ≠ 1) in order.
The artificial receptor may comprise an additional signal generating moiety in addition to the first signal generating moiety.
The artificial receptor may comprise more than two additional signal generating moieties.
The additional signal generating moiety may be a structure in which immune response signals of 4-1BB, CD27, CD28, ICOS and OX40 or other signals may be generated.
The conditions under which the additional signal generating portion generates an immune response signal and the characteristics of the immune response signal generated thereby include details corresponding to the immune response signal of the first signal generating portion and/or the second signal generating portion.
The immune response signal may be a signal that promotes cytokine synthesis, the immune response signal may be a signal that promotes or inhibits cytokine secretion, in particular, the cytokine may preferably be IL-2, TNF α, or IFN- γ.
The immune response signal may be a signal that aids in the growth or differentiation of other immune cells.
The immune response signal may be a signal that attracts other immune cells to the location where the signal appears.
The present invention encompasses all possible binding relationships for artificial receptors. Accordingly, aspects of the artificial receptors of the present invention are not limited to those described herein.
The artificial receptor may consist of an antigen recognition moiety-a receptor body-a first signal generating moiety. The acceptor body may optionally be included.
The artificial receptor may consist of an antigen recognition moiety-a receptor body-a second signal generating moiety-a first signal generating moiety. The acceptor body may optionally be included. In particular, the positions of the first signal generating section and the second signal generating section may be changed.
The artificial receptor may be composed of an antigen recognition moiety-a receptor body-a second signal generating moiety-a third signal generating moiety-a first signal generating moiety. The acceptor body may optionally be included. In particular, the positions of the first to third signal generating portions may be changed.
In the artificial receptor, the number of signal-generating moieties is not limited to 1 to 3, and may include more than 3.
In addition to the above embodiments, the artificial receptor may have a structure of an antigen recognition portion-a signal generating portion-a receptor body. This structure may be advantageous when it is desired to generate an immune response signal that acts outside the cell with the artificial receptor.
Artificial receptors can function in a manner comparable to wild-type receptors.
The artificial receptor can function to form a specific positional relationship by binding to a specific antigen.
The artificial receptor may function to recognize an antigen and generate an immune response signal that promotes an immune response against the particular antigen.
The artificial receptor may function to recognize an antigen of a general cell in vivo and suppress an immune response against the cell in vivo.
(iv) Signal sequence
In embodiments, the artificial receptor may optionally comprise a signal sequence.
This may help the artificial receptor to be easily localized on the membrane of the immune cell when the artificial receptor comprises a signal sequence of a specific protein. Preferably, when the artificial receptor comprises a signal sequence of a transmembrane protein, this may assist the artificial receptor in being located on the outer membrane of an immune cell across the membrane of the immune cell.
The artificial receptor may comprise one or more signal sequences.
The signal sequence may comprise a plurality of positively charged amino acids.
The signal sequence may comprise positively charged amino acids at positions near the N-terminus or C-terminus.
The signal sequence may be that of a transmembrane protein.
The signal sequence may be a signal sequence of a protein located on the outer membrane of an immune cell.
The signal sequence may preferably be that of an scFv.
The signal sequence may be included in the structure possessed by the artificial receptor, i.e., the antigen-recognizing portion, the receptor body, the first signal-generating portion, and the additional signal-generating portion.
In particular, the signal sequence may be located near the N-terminus or C-terminus of each structure.
In particular, the signal sequence may be about 100 amino acids from the N-terminus or C-terminus.
In embodiments, the artificial receptor may be a Chimeric Antigen Receptor (CAR).
The chimeric antigen receptor may be a receptor having binding specificity for one or more antigens.
The one or more antigens may be antigens specifically expressed by cancer cells and/or viruses.
The one or more antigens may be tumor associated antigens.
The one or more antigens may be, but are not limited to, human tumor receptor-inducing factors such as A, ALK, alpha-fetoprotein (AFP), adrenoreceptor 3 (ADRB), folate receptor, AD034, AKT, BCMA, -human chorionic gonadotropin, B7H (CD276), BST, BRAP, CD79, CD123, CD138, CD160, CD171, CD179, Carbonic Anhydrase IX (CAIX), CA-125, carcinoembryonic antigen (CEA), CCR, C-like molecule (CLL-1 or CLECL), claudin (NY), CXORF, CAGE, CDX, CLP, CT-7, HOCT/HOGE-TES-85, cTARGB, Epidermal Growth Factor Receptor (EGFR), EGFR type III (EGFRIN III), adhesion factor (NYNYNYNY), epithelial cell receptor (MAG-7), HPV-receptor (MAG-7, MAG-receptor), VEGF-receptor-2-receptor (MAG-7, MAG-receptor-2-receptor), VEGF-7, MAG-receptor-2-PEG-7, VEGF-2-PEG-7, VEGF-TNF-receptor-2-receptor-VEGF-2-VEGF-7, VEGF-7, VEGF-binding protein (MAG-7, VEGF-receptor-2-binding protein, VEGF-receptor-binding protein, VEGF-binding protein (MAG-receptor-binding protein, VEGF-7, VEGF-receptor-2-binding protein, VEGF-receptor-2-7, VEGF-binding protein, VEGF-receptor-binding protein, VEGF-7, VEGF-protein, VEGF-binding protein, VEGF-7, VEGF-2-protein, VEGF-2-receptor-binding protein, VEGF-2-protein, VEGF-receptor-7, VEGF-TNF-protein, VEGF-receptor-binding protein, VEGF-binding protein, VEGF-receptor-protein, VEGF-binding protein, VEGF-receptor-binding protein, VEGF-receptor-protein, VEGF-binding protein, VEGF-receptor-2-protein, VEGF-receptor-protein, VEGF-receptor-protein, VEGF-2-protein, VEGF-receptor-2-protein, VEGF-receptor-protein, VEGF-binding protein, VEGF-receptor-protein, VEGF-receptor-2-binding protein, VEGF-2-protein, VEGF-receptor-protein, VEGF-receptor-protein, VEGF-receptor-protein, VEGF-protein, VEGF-receptor-7, VEGF-2-protein, VEGF-7, VEGF-protein, VEGF-receptor-protein, VEGF-protein, human-protein, VEGF-receptor-protein, human-protein, VEGF-protein, human-receptor-protein, human-protein, VEGF-protein, human-protein-receptor-protein, human-protein.
As embodiments disclosed herein, the hybrid manipulated immune cell can be a human manipulated immune cell comprising all of:
(i) one or more artificially manipulated or modified immunomodulatory genes and/or expression products thereof; and
(ii) artificial receptor proteins and/or nucleic acids encoding same
The explanation for the above-mentioned one or more artificially manipulated or modified immunomodulatory genes is as described above.
(i) The expression product of (a) may be mRNA or protein expressed from one or more artificially manipulated or modified immunomodulatory genes.
Further, explanations regarding the above artificial receptors are as described above.
For example, the hybrid manipulated immune cell can be a functionally manipulated immune cell comprising an artificial receptor.
Here, the artificial receptor may be a chimeric antigen receptor, and explanations related thereto are as described above.
The mixed-manipulated immune cells can be immune cells having one or more artificially manipulated or modified immune modulatory genes, including one or more chimeric antigen receptors. Here, the immune cell having one or more immune regulatory genes which are artificially manipulated or modified may be a functionally manipulated immune cell, and explanations related thereto are as described above.
The immune cells that are manipulated in a mixed fashion can be immune cells in which expression of one or more immunomodulatory genes (including one or more chimeric antigen receptors) is repressed or inhibited. Here, the immune cell in which the expression of one or more immune regulatory genes is repressed or inhibited may be a functionally manipulated immune cell, as explained above in connection therewith.
As an example, a hybrid manipulated immune cell may be an immune cell produced by the artificial introduction of one or more nucleic acids or genes encoding a chimeric antigen receptor into a functionally manipulated immune cell.
Here, the nucleic acid or gene encoding the chimeric antigen receptor may be present in the cell in a form that is not inserted into the genome of the functionally manipulated immune cell.
Here, a nucleic acid or gene encoding a chimeric antigen receptor can be inserted into a specific locus of the genome of a functionally manipulated immune cell. The specific locus may be an intron, exon, promoter or enhancer locus of an immunomodulatory gene.
Here, the nucleic acid or gene encoding the chimeric antigen receptor can be inserted at will into one or more introns present in the genome of the functionally manipulated immune cell.
Here, the nucleic acid or gene encoding the chimeric antigen receptor can be inserted at will into one or more exons present in the genome of a functionally manipulated immune cell.
Here, the nucleic acid or gene encoding the chimeric antigen receptor can be inserted at will into one or more promoters present in the genome of the functionally manipulated immune cells.
Here, the nucleic acid or gene encoding the chimeric antigen receptor can be inserted at will into one or more enhancers present in the genome of the functionally manipulated immune cell.
Here, the nucleic acid or gene encoding the chimeric antigen receptor may be inserted at will into one or more regions other than introns, exons, promoters and enhancers present in the genome of the functionally manipulated immune cell.
The chimeric antigen receptor artificially introduced into the functionally manipulated immune cell may be expressed in the form of a protein in the hybrid manipulated immune cell, and the chimeric antigen receptor expressed as the protein may be located on the surface of the hybrid manipulated immune cell. Here, the functionally manipulated immune cells into which the nucleic acid or gene encoding the chimeric antigen receptor is artificially introduced may be in the form of hybrid-manipulated immune cells.
In another example, the hybrid manipulated immune cells can be immune cells generated by artificially introducing one or more chimeric antigen receptor proteins into functionally manipulated immune cells.
Chimeric antigen receptor proteins that are artificially introduced into functionally manipulated immune cells may be located on the surface of immune cells that are manipulated in a mixed fashion. Here, the functionally manipulated immune cells into which the chimeric antigen receptor protein is artificially introduced may be in the form of hybrid-manipulated immune cells.
In another example, the hybrid manipulated immune cells can be supplemented artificial structures of immune cells comprising one or more artificial manipulated or modified immune modulatory genes.
Here, the immune cells that supplement the artificial structures may be immune cells that comprise artificial receptors. The artificial receptor may be a chimeric antigen receptor.
Immune cells comprising one or more artificially manipulated or modified immune modulatory genes that supplement the artificial structure may have suppressed or suppressed expression of immune modulatory factors. Here, the immune cells that supplement the artificial structure in which one or more immunoregulatory genes are artificially modified may be in the form of hybrid manipulated immune cells.
In yet another example, the mixedly manipulated immune cells can be supplementary artificial structures of immune cells in which expression of one or more immunomodulatory factors is repressed or inhibited.
Aspects disclosed herein relate to methods of producing manipulated immune cells.
For explanations concerning the artificially modified immunoregulatory genes, reference is made to the explanations above. The method will be explained below focusing on a representative embodiment of the manipulated immune cells.
As an example, the method for producing a manipulated immune cell can be a method of producing a functionally manipulated immune cell. The method may be performed in vivo, ex vivo or in vitro.
In some embodiments, the method comprises: the method comprises the steps of sampling cells or cell groups from human or non-human animals, and modifying the cells or cell groups. The culture may be carried out at any step ex vivo. The cells may even be reintroduced into a non-human animal or plant.
In embodiments, the method may be a method for generating a functionally manipulated immune cell comprising one or more artificially manipulated immunoregulatory genes, the method comprising contacting:
(a) an immune cell;
(b) a composition for gene manipulation capable of manual manipulation of at least one immunomodulatory gene selected from the group consisting of: PD-1 gene, CTLA-4 gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene, PPP2r2d, TET2 gene, PSGL-1 gene, A20 gene and KDM6A gene.
Here, (a) the immune cell may be isolated from a human body, or may be an immune cell differentiated from a stem cell.
(b) Compositions for gene manipulation comprise the following:
(b') a guide nucleic acid that hybridizes to the target sequence of SEQ ID NO: 1-SEQ ID NO: 289 have homology or are capable of forming complementary binding therewith: PD-1 gene, CTLA-4 gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene, PPP2r2d gene, TET2 gene, PSGL-1 gene, A20 gene and KDM6A gene; and
(b ") an editing protein, which is one or more proteins selected from the group consisting of: a Cas9 protein derived from streptococcus pyogenes, a Cas9 protein derived from campylobacter jejuni, a Cas9 protein derived from streptococcus thermophilus, a Cas9 protein derived from staphylococcus aureus, a Cas9 protein derived from neisseria meningitidis, and a Cpf1 protein.
The explanation about the above-mentioned composition for gene manipulation is as described above.
The contacting can be performed ex vivo.
The contacting may comprise introducing (b) a composition for gene manipulation into (a) an immune cell.
The method may be performed in vivo or in vitro (e.g., ex vivo).
For example, the contacting can be performed in vitro, and the contacted cells can be returned to the subject after contacting.
The method may use immune cells in vivo or isolated from a living body (e.g., a human body), or artificially produced immune cells. As an example, contacting a cell from a subject having cancer may be included.
The immune cells used in the methods can be those derived from mammals including primates (e.g., humans, monkeys, etc.) and rodents (e.g., mice, rats, etc.). For example, the immune cell can be an NKT cell, an NK cell, a T cell, and the like. Here, the immune cell may be a manipulated immune cell that is supplemented with an immune receptor (e.g., a Chimeric Antibody Receptor (CAR) or a manipulated T Cell Receptor (TCR)).
The method may be carried out in a medium suitable for immune cells, which may contain serum (e.g., fetal bovine serum or human serum), interleukin-2 (IL-2), insulin, IFN- γ, IL-4, IL-7, GM-CSF, IL-10, IL-15, TGF- β, and TNF- α, or a suitable medium may contain factors necessary for proliferation and survival, including other cell growth additives known to those skilled in the art (e.g., minimal essential medium, RPMI medium, or X-vivo 1640-10, X-vivo-15, X-vivo-20(Lonza)), but the medium is not limited thereto.
In another example, the method of producing a manipulated immune cell can be a method of producing a mixed-type manipulated immune cell. The method may be performed in vivo, ex vivo or in vitro.
In some embodiments, the method comprises: the method comprises the steps of sampling cells or cell groups from human or non-human animals, and modifying the cells or cell groups. The culture may be carried out at any step ex vivo. The cells may even be reintroduced into a non-human animal or plant.
In embodiments, the method may be a method for producing a hybrid manipulated immune cell comprising one or more human manipulated immunoregulatory genes and one or more artificial receptors, the method comprising contacting:
(a) an immune cell;
(b) compositions or artificial receptor proteins for artificial receptor expression; and
(c) a composition for gene manipulation capable of manual manipulation of one or more immunomodulatory genes selected from the group consisting of: PD-1 gene, CTLA-4 gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene, PPP2r2d, TET2 gene, PSGL-1 gene, A20 gene and KDM6A gene.
(a) The immune cells may be isolated from a human body, or may be immune cells differentiated from stem cells.
(b) The composition for artificial receptor expression may be a composition comprising a vector having a nucleotide sequence encoding a chimeric antigen receptor.
The composition for artificial receptor expression of (b) may be introduced into immune cells using one or more methods selected from the group consisting of electroporation, liposome, plasmid, viral vector, nanoparticle, and protein translocation domain PTD) fusion protein method.
For example, the viral vector may be one or more selected from the group consisting of retrovirus, lentivirus, adenovirus, adeno-associated virus (AAV), vaccinia virus, poxvirus, or herpes simplex virus.
(c) Compositions for gene manipulation may comprise the following:
(c') a guide nucleic acid that hybridizes to the target sequence of SEQ ID NO: 1-SEQ ID NO: 289 have homology or are capable of forming complementary binding therewith: PD-1 gene, CTLA-4 gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene, PPP2r2d gene, TET2 gene, PSGL-1 gene, A20 gene and KDM6A gene; and
(c ") an editing protein, which is one or more proteins selected from the group consisting of: a Cas9 protein derived from streptococcus pyogenes, a Cas9 protein derived from campylobacter jejuni, a Cas9 protein derived from streptococcus thermophilus, a Cas9 protein derived from staphylococcus aureus, a Cas9 protein derived from neisseria meningitidis, and a Cpf1 protein.
The explanation about the above-mentioned composition for gene manipulation is as described above.
The contacting may be performed ex vivo.
The contacting may be sequential or simultaneous with (a) the immune cell and (b) the composition for artificial receptor expression and (c) the composition for gene manipulation.
The contacting may be sequential or simultaneous with (a) an immune cell and (c) a composition for gene manipulation and (b) a composition for artificial receptor expression.
The contacting may comprise introducing (b) a composition for artificial receptor expression and (c) a composition for gene manipulation into (a) an immune cell.
The method may be performed in vivo or ex vivo (e.g., in vitro).
For example, the contacting can be performed ex vivo, and the contacted cells can be returned to the subject after contacting.
The methods can employ immune cells in an organism or an organism, such as immune cells isolated from a human or artificially produced immune cells. In one example, contacting a cell from a subject having cancer can be included.
The immune cells used in the above methods can be derived from mammals including primates (e.g., humans, monkeys, etc.) and rodents (e.g., mice, rats, etc.). For example, the immune cell can be an NKT cell, an NK cell, a T cell, and the like. In particular, the immune cell may be a manipulated immune cell that is supplemented with an immune receptor (e.g., a Chimeric Antibody Receptor (CAR) or a manipulated T Cell Receptor (TCR)).
The method may be carried out in a medium suitable for immune cells, which may contain serum (e.g., fetal bovine serum or human serum), interleukin-2 (IL-2), insulin, IFN- γ, IL-4, IL-7, GM-CSF, IL-10, IL-15, TGF- β, and TNF- α, or a suitable medium may contain factors necessary for proliferation and survival, including other cell growth additives known to those skilled in the art (e.g., minimal essential medium, RPMI medium, or X-vivo 1640-10, X-vivo-15, X-vivo-20(Lonza)), but the medium is not limited thereto.
Aspects disclosed herein relate to methods of treating diseases using manipulated immune cells.
Embodiments disclosed herein are uses for the treatment of disease using immunotherapy methods that include administering artificially modified cells (e.g., genetically modified immune cells containing a chimeric antigen receptor) to a subject.
The subject to be treated can be a mammal including primates (e.g., humans, monkeys, etc.) and rodents (e.g., mice, rats, etc.).
Embodiments disclosed herein are pharmaceutical compositions for the treatment of disease using artificially modified immune cells.
The pharmaceutical composition may be used in the treatment of diseases using an immune response. For example, the pharmaceutical composition is a composition containing modified immune cells. The pharmaceutical composition may be referred to as a composition for treatment or a cell therapy product.
In one example, the pharmaceutical composition may comprise a functionally modified immune cell.
Here, the functionally modified immune cell population comprised in the pharmaceutical composition may represent 50-60%, 60-70%, 70-80%, 80-90% or 90-100% of the total immune cell population comprised in the pharmaceutical composition.
In another example, the pharmaceutical composition may comprise a mixed-mode manipulated immune cell.
Here, the functionally modified immune cell population comprised in the pharmaceutical composition may represent 50-60%, 60-70%, 70-80%, 80-90% or 90-100% of the total immune cell population comprised in the pharmaceutical composition.
In yet another example, the pharmaceutical composition can comprise a functionally manipulated immune cell and a mixedly manipulated immune cell.
Here, the functionally modified immune cell population comprised in the pharmaceutical composition may represent 1-20%, 20-60%, 60-80% or 80-99% of the total immune cell population comprised in the pharmaceutical composition; in this case, the mixed type modified immune cell population included in the pharmaceutical composition may account for 80% -99%, 60% -80%, 40% -60%, 20% -40%, or 1% -20% of the total immune cell population included in the pharmaceutical composition.
Here, the pharmaceutical composition may further comprise additional components.
For example, the pharmaceutical composition may comprise an immune checkpoint inhibitor.
Here, the immune checkpoint inhibitor may be an inhibitor of PD-1, PD-L1, LAG-3, TIM-3, CTLA-4, TIGIT, BTLA, IDO, VISTA, ICOS, KIR, CD160, CD244, or CD 39. Here, the inhibitor may be, but is not limited to: an antibody; a compound; a nucleic acid, peptide, polypeptide or protein capable of binding to or interacting with an immune checkpoint; microrna (mirna), small interfering RNA (sirna), or short hairpin RNA (shrna) for RNA interference (RNAi); nucleases for the knock-out or knockdown of immune checkpoint genes, such as Zinc Finger Nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), or CRISPR/Cas.
The pharmaceutical composition may comprise an antigen binding vehicle.
The pharmaceutical composition may comprise a cytokine.
The pharmaceutical composition may comprise a cytokine secretion stimulator or repressor.
The pharmaceutical composition may comprise a suitable carrier for delivering the manipulated immune cells into the body.
Here, the immune cells included in the pharmaceutical composition may be autologous cells of the patient or allogeneic cells of the patient.
Another embodiment disclosed herein is a method of treating a disease in a patient, comprising producing a pharmaceutical composition as set forth above and administering the pharmaceutical composition to a patient in need thereof.
The disease to be treated
The disease may be an immune disease.
In particular, the immune disease may be a disease in which the immune competence (immunity competition) is deteriorated.
The immune disease may be an autoimmune disease.
For example, autoimmune diseases can include Graft Versus Host Disease (GVHD), systemic lupus erythematosus, celiac disease, type I diabetes, graves' disease, inflammatory bowel disease, psoriasis, rheumatoid arthritis, multiple sclerosis, and the like.
In addition, the disease may be a refractory disease whose pathogen is known but whose treatment is unknown.
The intractable disease may be a viral infection disease.
The refractory disease can be a disease caused by a prion pathogen.
The refractory disease can be cancer.
Treatment for enhancing immunity
For patients with significant reductions in immunity, even mild infections can lead to fatal outcomes. The decreased immunity is caused by decreased immune cell function, decreased immune cell yield, etc. As a method for enhancing immunity to treat immune function deterioration, there are a permanent treatment method of activating the production of normal immune cells and a temporary treatment method of performing transient injection of immune cells.
An immunity-enhancing treatment may be intended to permanently enhance immunity by injecting a therapeutic composition into the patient.
The treatment to enhance immunity may be a method of injecting a therapeutic composition into a specific body part of a patient. In particular, the specific body part may be a part having tissue that supplies a source of immune cells.
The treatment to enhance immunity may be to create a new source of immune cells in the patient. In particular, in one example, the therapeutic composition can comprise stem cells. In particular, the stem cells may be hematopoietic stem cells.
An immune enhancing treatment may be intended to inject a therapeutic composition into a patient to temporarily enhance immunity.
The treatment to enhance immunity may be by injecting a therapeutic composition into the patient.
In particular, preferred therapeutic compositions may contain differentiated immune cells.
Therapeutic compositions for treatments that enhance immunity may contain a specific number of immune cells.
The specific amount may be changed according to the degree of deterioration of immunity.
The specific number may be varied according to the body volume.
The specific amount may be adjusted according to the amount of cytokine released by the patient.
Treatment of refractory diseases
Immune cell manipulation techniques can provide a therapeutic approach to diseases against pathogens such as HIV, prions, and cancer, which are not yet fully treated. Although the pathogens of these diseases are known, in many cases, it is difficult to treat these diseases due to the problems of difficulty in forming antibodies, rapid development of the disease, inactivation of the immune system of the patient, and latency of the pathogens in the body. Manipulated immune cells can be a powerful means to address these problems.
Treatment of refractory diseases can be carried out by injecting the therapeutic composition into the body. In particular, preferred therapeutic compositions may contain manipulated immune cells. In addition, the therapeutic composition may be injected into a particular body part.
The manipulated immune cells can be immune cells with improved recognition of the pathogen of the disease of interest.
The manipulated immune cells may be immune cells with enhanced strength or activity of the immune response.
-gene correction therapy
In addition to a therapeutic method by means of immune cells extracted from an external source, there is a therapeutic method in which the expression of immune cells is directly influenced by manipulating genes of a living body. The treatment method can be carried out by directly injecting a gene modification composition for gene manipulation into the body.
The gene modification composition may contain a guide nucleic acid-editing protein complex.
The gene modification composition may be injected into a particular body part.
The particular body part may be a source of immune cells, such as bone marrow.
The subject to be administered may be a mammal including primates (e.g., humans, monkeys, etc.) and rodents (e.g., mice, rats, etc.).
Administration of the composition may be effected by any convenient means, e.g., injection, infusion, implantation, transplantation, etc. The route of administration may be selected from subcutaneous, intradermal, intratumoral, intranodal, intramedullary, intramuscular, intravenous, intralymphatic, intraperitoneal and the like.
A single dose of the composition (a pharmaceutically effective amount to achieve the desired effect) may be selected from about 104-109The whole integer number (e.g., about 10) in the range of individual cells/kg body weight of the subject to be administered5-106Individual cells/kg body weight), but the dose is not limited thereto; the age, health and weight of the subject to be administered may be considered; the nature of concurrent therapy, if any, frequency of treatment and the desired effect, to properly prescribe a single dose of the composition.
For example, when immune modulatory genes are manually manipulated and controlled by the compositions for gene manipulation disclosed herein, the immune potency involved in the survival, proliferation, persistence, cytotoxicity, cytokine release and/or infiltration of immune cells can be improved. In addition, immune diseases and intractable diseases can be alleviated or cured by a pharmaceutical composition comprising the manipulated immune cells disclosed in the present specification and a treatment method using the pharmaceutical composition.
Examples
Design of sgRNA
CRISPR/Cas9 target regions of human PD-1 gene (PDCD 1; NCBI accession No. NM _005018.2), CTLA-4 gene (NCBI accession No. NM _001037631.2), a20 gene (TNFAIP 3; NCBI accession No. NM _001270507.1), DGK α gene (NCBI accession No. NM _001345.4), DGK ζ gene (NCBI accession No. NM _001105540.1), EGR2 gene (NCBI accession No. NM _000399.4), PPP2r2d gene (NCBI accession No. NM _001291310.1), PSGL-1 gene (NCBI accession No. NM _001193538.1), TET2 gene (NCBI accession No. NM _017628.4) were selected using CRISPR RGEN tool (Institute for Basic Science, korea) and evaluated by off-target test, target regions of CRISPR/Cas9 with target regions of CRISPR/Cas 42, target regions with no target regions other than the target regions of 0, 1bp or no target regions were selected as target DNA of sgr 2 bp.
Synthesis of sgRNA
PCR amplification of the sgRNA synthetic template was performed by annealing and extension of two complementary oligonucleotides.
The target region sequences used at this time, the primer sequences used for amplifying them, and the DNA target sequences targeted by the sgrnas obtained therefrom are described in table 2 below.
In vitro transcription of the template DNA ('NGG' with 3 'removal of the target sequence) was performed using T7 RNA polymerase (New England Biolabs), RNA was synthesized according to the manufacturer's instructions, and the template DNA was subsequently removed using DNAse (Ambion). The transcribed RNA was purified using the Expin Combo kit (GeneAll) and isopropanol precipitation.
In experiments using T cells, to minimize immunogenicity and degradation of sgrnas, alkaline phosphatase (New England Biolabs) was used to remove 5' terminal phosphate residues from sgrnas synthesized using the above method, followed by repurification of RNA using the expincobo kit (geneale) and isopropanol precipitation. Furthermore, chemically synthesized sgrna (trilink) was used in some T cell experiments.
Chemically synthesized sgrnas used in certain embodiments are sgrnas with 2' OMe and phosphorothioate modifications.
For example, DGK α sgRNA #11 used in this example has
Figure BDA0002359848460001571
Figure BDA0002359848460001572
(2'OMe ═ 2' -methyl RNA; ps ═ phosphorothioate) structure.
In another example, a20 sgRNA #1 used in this example is
Figure BDA0002359848460001573
Figure BDA0002359848460001574
(the bold part is a sequence hybridized with the target sequence region; sgRNA for other target genes or other target sequences has the bold sequence as the target sequence (except T to U), or the above sequence may be modified in which 2' -OMe modification and phosphorothioate skeleton introduction are performed to three nucleotides at the 3' end and three nucleotides at the 5' end of the sequence.
[ Table 2]
Figure BDA0002359848460001581
Figure BDA0002359848460001591
Figure BDA0002359848460001601
Figure BDA0002359848460001611
Figure BDA0002359848460001621
Figure BDA0002359848460001631
Figure BDA0002359848460001641
Figure BDA0002359848460001651
Figure BDA0002359848460001661
Figure BDA0002359848460001671
Figure BDA0002359848460001681
Figure BDA0002359848460001691
Figure BDA0002359848460001701
Figure BDA0002359848460001711
Figure BDA0002359848460001721
Figure BDA0002359848460001731
Figure BDA0002359848460001741
Figure BDA0002359848460001751
Figure BDA0002359848460001761
Figure BDA0002359848460001771
Figure BDA0002359848460001781
Figure BDA0002359848460001791
Figure BDA0002359848460001801
Figure BDA0002359848460001811
Figure BDA0002359848460001821
Figure BDA0002359848460001831
Figure BDA0002359848460001841
Figure BDA0002359848460001851
Figure BDA0002359848460001861
Figure BDA0002359848460001871
Figure BDA0002359848460001881
Figure BDA0002359848460001891
Figure BDA0002359848460001901
Figure BDA0002359848460001911
Figure BDA0002359848460001921
Figure BDA0002359848460001931
Meanwhile, with respect to the DNA target sequences depicted in table 1, SEQ ID NOs: 1-SEQ ID NO: 84, sgRNA was produced in the same manner as described above for the PSGL-1 gene.
Example 3 selection of sgRNAs in Jurkat cells
The activity of the above synthetic sgrnas targeting exons of a20, DGK α, EGR2, PPP2r2d, PD-1, CTLA-4, DGK ζ, PSGL-1, KDM6A, and TET2 were tested in Jurkat cells.
Jurkat cells (ATCC TIB-152; immortalized cell line of human T cells) were cultured in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum (GeneAll). And at 37 ℃ and 5% CO2The cells were cultured in an incubator under the conditions of (1).
For activating the cells, the cell concentration in the medium was maintained at 1X 106Individual cells/mL.
CD2/CD3/CD28 beads (anti-CD 2/3/CD28 Dynabeads; Miltenyi Biotec) were added at a ratio of 3:1 (beads: cells; based on the number of beads and cells) and at 37 ℃ and 5% CO2The cells were cultured in an incubator under the conditions of (1). After 72 hours of cell activation, the CD2/CD3/CD28 beads were removed with a magnet and the cells were further cultured for 12-24 hours under a beadless condition.
Mu.g of in vitro transcribed sgRNA and 4. mu.g of Cas9 protein (ToolGen) were introduced by electroporation into 1X 106Cultured Jurkat cells (in vitro). Genes were introduced using a 10. mu.L tip of a Neon transfection system (ThermoFisher Scientific, Grand Island, NY) under the following conditions:
jurkat (buffer R): 1,400V, 20ms, 2 pulses.
Cells were plated on 500. mu.L antibiotic-free medium at 37DEG C and 5% CO2The culture was performed in an incubator.
The proportion of insertions deleted was tested on transfected Jurkat cells (designated "+ aRGEN") compared to untransfected Jurkat cells (designated "+ aRGEN"). The CRISPR/Cas9 target sequences tested are summarized in table 3, and the indel ratios for the various sgrnas are summarized in table 4.
[ Table 3]
Figure BDA0002359848460001951
Figure BDA0002359848460001961
Figure BDA0002359848460001971
Figure BDA0002359848460001981
Figure BDA0002359848460001991
Figure BDA0002359848460002001
Figure BDA0002359848460002011
Figure BDA0002359848460002021
Figure BDA0002359848460002031
Figure BDA0002359848460002041
[ Table 4] Activity of each sgRNA on target sequences on Jurkat cells
Figure BDA0002359848460002051
Figure BDA0002359848460002061
Figure BDA0002359848460002071
Figure BDA0002359848460002081
Figure BDA0002359848460002091
Figure BDA0002359848460002101
Figure BDA0002359848460002111
Figure BDA0002359848460002121
4. Tumor cell line culture
EGFRvIII positive U87 MG glioblastoma cell line (U87vIII) was purchased from Celther Polska. A375P melanoma Cell line was purchased from Korean Cell line Bank. The cell lines were cultured in DMEM medium containing 10% fetal bovine serum albumin (FBS).
5. Lentiviral preparation
From the study of Sampson, Choi et al (Sampson et al, 2014, Rapoport, Stadtmauer et al, 2015) reference was made to an anti-EGFRVIII scFV fused to a fusion protein 139CAR containing a CD8 hinge, 4-1BB and a CD3 zeta domain, and a c259TCR construct targeting NY-ESO-1. Codon optimized cDNA CAR and TCR constructs were subcloned with pLVX vector. Lentivirus vectors and helper plasmids were transfected into 293T cells using Lipofectamine2000(ThermoFisher SCIENTIFIC), and culture supernatants of the produced lentiviruses were obtained by culturing the transfected 293T cells. After obtaining the culture supernatant, the culture supernatant containing lentivirus was overlaid at a ratio of 4:1 to a sucrose-containing buffer (100mM NaCl, 0.5mM ethylenediaminetetraacetic acid [ EDTA ], 50mM Tri-HCl, pH 7.4) and centrifuged at 10,000g for 4 hours at 4 ℃. After centrifugation, the supernatant was removed and resuspended after addition of Phosphate Buffered Saline (PBS).
Construction of DGK KO 139CAR-T cells
Human peripheral blood T cells (pan-T cells) were purchased from STEMCELL TECHNOLOGIES. Thawed T cells were cultured overnight in RPMI medium supplemented with FBS, 50U/mL hIL-2, and 5ng/mL hIL-7 prior to activation. anti-CD 3/CD28 Dynabeads (ThermoFisher scientific) were used to activate cells in RPMI medium supplemented with 10% FBS at a ratio of 3:1 (beads: cells). After 24 hours of activation, T cells were mixed with 139-CAR lentivirus in 100. mu.g/mL retronectin coated plates for 48 hours. Beads were removed after 3 days of stimulation. Electroporation was performed using Amaxa P3 primary cell kit and 4D-nucleofecter (Lonza). To form the Cas9 Ribonucleoprotein (RNP) complex, 40 μ g of recombinant streptococcus pyogenes Cas9(Toolgen) was incubated with 10 μ g of chemically synthesized tracr/crrna (integrated DNA technologies) for 20 min. Pre-incubated Cas9 RNP complex was added to 3 × 10 resuspended in P3 buffer6In individual stimulated T cells. The Cas9 RNP complex was introduced into the nucleus using the program EO-115. After electroporation, cells were plated at 5X 105The concentration of individual cells/mL was inoculated in RPMI medium supplemented with 50U/mL hIL-2, 5ng/mL hIL-7 and 10% FBS the target sequences of the crRNA used in the assay were DGK α: CTCTCAAGCTGAGTGGGTCC and DGK ζ: ACGAGCACTCACCAGCATCC.
7. Flow cytometry staining and antibodies
Unless otherwise indicated, cell staining was performed in PBS with 1% FBS added at 4 ℃. The list of antibodies and reagents used for flow cytometry and functional studies is as follows: CellTrace CFSE/Far red (ThermoFisher); 7-AAD (Sigma); anti-CD 3: UCHT1 (BD); anti-CD 4: RPA-T4 (BD); anti-CD 8: HIT8a (BD); anti-CD 56: b159 (BD); anti-NKG 2D: 1D11 (Biolegend); anti-CD 45 RO: UCHL1 (BD); anti-CCR 7: 150513 (BD); anti-PD-1: EH12.2H7 (Biolegend); anti-CD 25: M-A251 (BD); anti-Fas: dx2 (BD); anti-CD 107 a: h4a3(Molecular Probes); anti-EGFRviii: (Biorbyt); goat anti-human IgG: (Biorad). Data were collected in an Attune NxT sonic focused cytometer and analyzed using flowjo.
8. In vitro killing assay, cytokine release and proliferation assay
U87vIII and A375P were stained with CellTrace Far red (Invitrogen). Tumor cell lines were co-cultured with T cells at the indicated ratios and distributed 2X 10 per well in U-bottom 96-well plates4To 5X 104And (3) a tumor cell line. Resting 139CAR-T cells and c259T cells were added to each target cell at the indicated effector to target (E: T) ratio. Cells were harvested after 18 hours of co-culture and stained with 7-amino actinomycin to identify live/dead cells. The samples were measured with an Attune NxT sonic focused cytometer and analyzed with FlowJo. Equation [ (lysis sample value% -lysis minimum%)/(lysis maximum% [ 100%]-minimum% cleavage)]X 100%) to calculate cytotoxicity. The test was repeated 3 times. The culture supernatant collected after co-culture was used to measure the amount of secretion of IL-2 and IFN-. gamma.using ELISA kit (Biolegend). Celltrace-labeled 139CAR-T cells were co-cultured with U87vIII cells for 4 days for proliferation analysis and the distribution of Celltrace in 139CAR-T cells was evaluated using flow cytometry.
9. Repeated tumor challenge experiment
For continuous tumor testing, 139CAR-T was co-cultured with U87vIII in IL-7 medium at a ratio of 3:1(E: T) (day 0). On day 4, 139CAR-T cells were harvested and co-cultured again with U87vIII in IL-7 medium at the same E: T ratio. Culture supernatants were collected 24 hours after primary and secondary tumor inoculation to evaluate IFN-. gamma.and IL-2 release, respectively.
10. Western blot analysis
To evaluate the TCR distal signal of T cells, 1X 10 was applied by using anti-CD 3 activated beads (Miltenyi Biotec) at a ratio of 1:2 (beads: cells)6Individual cells were activated for 15 min and 60 min. To measure ERK, pERK, and GAPDH, cell lysates were prepared using RIPA lysis and extraction buffer. Each antibody used in the assay was purchased from Cell Signaling.
11. Calcium influx
The measurement of the calcium influx into T cells was performed according to the manual of calcium assay kits (BD). Briefly, T cells were washed with RPMI medium, resuspended in the same medium, and incubated with the stain for 1 hour at 37 ℃. After obtaining the basic standard for FITC signal from non-treated cells, anti-CD 3 activated beads (Miltenyi Biotec) were added at a bead to cell ratio of 5:1 and measured by flow cytometry. Data collected from flow cytometry was analyzed using kinetic mode with FlowJo software.
12. Real-time PCR
For RNA sequencing, human T activator anti-CD 3/CD28 Dynabeads (Thermofeisher) was used to mix 1X 10 at a ratio of 1:1 (beads: cells)6The cells were activated for 48 hours RNA was extracted using RNeasy Mini kit (Qiagen) and cDNA was produced according to manufacturer's instructions (ABI), real-time PCR was performed using TaqMan gene expression analysis kit/probe set (Thermofish), expression of each gene was normalized with GAPDH expression here, the primers used for the test were as follows, hDGK α F: 5'-AATACCTGGATTGGGATGTGTCT-3', hDGK α R: 5' -GTCCGTCGTCCTTCAGAGTC, hDGK ζ F: 5'-GTACTGGCAACGACTTGGC-3', hDGK ζ R: 5' -GCCCAGGCTGAAGTAGTTGTT, h β F: 5'-ggcactcttccagccttc-3', h β R: 5'-tacaggtctttgcggatgtc-3', ID 2: Hs00747379_ m1, PRDM 1: HS00153357_ m1, IL 10: 00174086_ m1, IFNG: Hs00174143_ m1, IL 2: HS 00174114.
13. Double genome sequencing
Genomic DNA of human T cells was isolated using DNeasy Tissue kit (Qiagen). Genomic DNA (20. mu.g) was treated with Cas9 protein (10. mu.g), crRNA (3.8. mu.g) and tracrRNA (3.8. mu.g) in 1000. mu.L of reaction solution (NEB3.1 buffer) and incubated at 37 ℃ for 4 hours. The digested DNA was incubated with RNase A (50. mu.g/mL) at 37 ℃ for 30 min and purified with DNeasy Tissue kit. The digested DNA was fragmented using the Covaris system and ligated with linkers to form a library. The DNA library was applied to the whole genome sequence using Illumina HiSeq × Ten sequence from thermogen ETEX. To form a Bam file, Isaac aligner is used, using the following parameters: version 01.14.03.12; human genome reference, hg19 from UCSC (original GRCh37 from NCBI, 2 months 2009); mouse genomic reference, mm10 from UCSC; base amount truncation, 15; keeping reading repeatedly, yes; variable read length support, yes; realign gaps, No; and joint shear, is (joint: 5'-AGATCGGAAGAGC-3', 5'-GCTCTTCCGATCT-3')
14. Mouse xenograft study
For the U87vIII tumor model, 1X 10 in 100. mu.L PBS volume6One U87vIII cell was injected subcutaneously into the right flank of a female NSC mouse at 6-8 weeks (day 0). On day 28 after transplantation, the tumor size reached 150. + -.50 mm2And mice were randomly grouped. Each group consisted of 6-8 mice and tumors were of similar size. Intravenous (IV) or Intratumoral (IT) injections of 5X 10 were administered to each group on day 28 and day 32, respectively6Identification of surface expression of CAR for 139AAVS1 CAR-T cells and 139DGK α ζ CAR-T cells (surface CAR expression range: 25% -70%). from day 32 to day 35, Temozolomide (TMZ) (Sigma) (0.33 mg/mouse/day) was administered intraperitoneally to each mouse daily3Mice were sacrificed at time. To further study CAR-T cells after in vivo delivery, peripheral blood, spleen and tumor tissue were isolated from each mouse. To isolate the tumor tissue, the tumor samples were trimmed with scissors and treated with 100U/ml collagenase IV and 20U/ml DNase in a37 ℃ water bath for 1 hour. The cells were then passed through a sterile cell filter for further study. To isolate cells from the spleen, the tissue was crushed with a sterile plunger and passed through a sterile cell filter. For hemolysis, ACK buffer (150mM NH)4Cl，10mM KHCO31mM EDTA, pH 7.2) for an additional 5 minutes to cell suspension. Is composed ofAnalysis of effector function of tumor infiltrating T cells in CO2Cells dissociated from tumor tissue were reactivated with 50ng/mL PMA and 1. mu.g/mL ionomycin in an incubator for 5 hours, and intracellular IFN-. gamma.and TNF α staining was performed.
Example 1 efficient inhibition of DGK in human primary T cells by optimized CRISPR/Cas9 RNP delivery
To identify the role of DGK in the anti-tumor activity of human primary T cells, grna targeting exon 5-exon 7 of the DGK α gene and exon 3-exon 12 of the DGK zeta gene were screened after optimized CRISPR/Cas9 RNP electroporation and lentiviral transfection, respectively, T cells treated with CRISPR/387cas 2 showed active CAR expression (fig. 28) even though a slight decrease in viability and cell growth was observed, viability and cell growth rapidly recovered 2 days after electroporation (fig. 29) in the example 139 (anti-EGFRvIII CAR with high specificity) was used to target glioblastoma cells (Sampson et al, 2014) the expression of EGFRvIII was strictly restricted to malignant tissue, thus concerns about potential safety (e.g. targeting effect) identified by manipulation of EGFRvIII using 139-CAR was improved based on the insertion deletion ratio in depth-determined single gene knockout experiment of about 80% -90% (fig. 30) CAR was found to be effective in the targeted knock-out of human cells by the engineered T cell knock-out gene analysis of the single gene expression of the dna knockout CAR (map 5-knock-out gene) and the results of the single gene expression of the double knockout cell mediated deletion map expression of the protein by the DGK-mediated knock-out gene expression of the DGK knock-down gene by the DGK 5-mediated map knock-out gene (fig. 9-out gene) showed significant loss-mediated by the comparative study of the results of the non-mediated knock-down gene expression of the non-mediated knock-down protein in the prior art study of the anti-T-mediated knock-cell (s 139-mediated by the study, the study of the full-T-mediated knock-T139-mediated gene expression of the study, the study of the full-T-cell, the study of the expression.
Example 2 enhanced effector function presented by CD3 terminal signals amplified by artificially manipulated DGK
Since increased cytokine secretion in DGK zeta-deficient T cells was previously reported by different groups, the antitumor function of DGK139 CAR-T cells was evaluated in the examples (Shin et al, 2012; Riese et al, 2013) in vitro profiling, AAVS 1139 CAR-T (which maintains 139CAR-T cytotoxicity and has a more similar physiological state than DGK139 CAR-T) was used as a negative control (fig. 32) when co-cultured with U87vIII DGK 139-T showed superior effector functions, such as a significant increase in cytokines and cytotoxicity (fig. 33) compared to AAVS1 CAR-T, interestingly DGK139 CAR-T, DGK α -T produced more IFN- γ and IL-2 than DGK α or DGK139 CAR-T, strongly suggesting that k double knockout had a synergistic effect in antitumor activity when CAR-T cells and tumor cell lines were co-cultured and this was also shown to block the effector functions (PD) expressed in DGK 1) when CAR-T cells were cultured with antibodies (CAR-1) expressed as a potent antagonist PD-1.
Next, we investigated whether the signal1(CD3 signal) pathway is disrupted by DGK.AAVS 1T cells and DGK T cells were stimulated with anti-CD 3 beads for the indicated time periods and calcium influx and ERK (peripheral signal to signal 1) were measured, calcium influx is not affected by TCR activation, but phosphorylated ERK signal is amplified and persists longer in DGK knock-out mutants (FIG. 35). the dramatic increase in phosphorylated ERK signal in DGK α zeta T cells is consistent with the synergy of DGK double knock-out in FIG. 33. this result demonstrates that removal of DGK increases TCR peripheral signal, thereby increasing T cell cytotoxicity and cytokine release.
Example 3 escape of the immunosuppressive effects of TGF- β and PGE2 by T cells manually manipulated through DGK.
Removal of DGK from previous examples found effective activation of TCR signaling, whether artificially manipulated DGK could reduce T cell sensitivity to signal1 inhibitors or not was tested since the therapeutic results of CAR-T treatment approaches were limited by high levels of TGF- β and PGE2 in the Tumor Microenvironment (TME), TGF- β and PGE2 were of major concern in the inhibitory factors (arumuugam, Bluemn et al, 2015; Perng and Lim (2015); O' rour, nasrallahh et al, 2017). first, studies were made on the inhibitory potency of TGF-37ζ 0 against TGF-T activity with respect to U87 vIII. CAR 139-T2 and TGF- β 1 mediated immunosuppression since DGK double knockout CAR showed synergistic resistance in PGE2 and TGF- β 1 mediated immunosuppression, CAR β ζ 139-T was used to test (figure 36) when exposure to high physiological concentrations of TGF- β (10 ng/CAR), CAR, cd- β, cd7 showed significant inhibition of intracellular apoptosis when the anti-TGK receptor activity was maintained as a decrease in pgk receptor response to TGF-T receptor 5, pg7-T receptor antagonist response to the anti-T receptor agonist therapy results of TGF-T receptor agonist therapy approaches showed no inhibition by the same, no inhibition effect as observed by afv β 5, No. 5, no inhibition of TGF-T receptor 5, no inhibition of TGF-T receptor activity, no inhibition of TGF-T receptor 5, No. 5, no inhibition of TGF-7, no inhibition of TGF-T receptor activity, No. 5, no inhibition of TGF-T receptor activity, No. 5 in the inhibition of TGF-T receptor 5, no inhibition of TGF-T receptor activity, No. 5, no inhibition of TGF-T receptor activity, No. 5, no inhibition of tumor activity, No. 5, No. was confirmed by tumor activity, No. 5 was shown to tumor activity, No. 5 was shown to inhibition of tumor activity in the inhibition of tumor activity in the inhibition of tumor activity.
Example 4 maintenance of effector function of DGK-manipulated T cells in repeated antigen stimulation
When repeatedly recognizing an antigen, T cells are often in an inhibitory state, where IL-2 secretion and cytotoxicity are lost DAG metabolism is an important determinant regulating T cell activation and zeta-deprivation (Olenchock, Guo et al, 2006; Zha, Marks et al, 2006). numerous studies report that pharmacological inhibition of DGK α can reverse the state of mouse T cell disability, thereby identifying whether human T cells artificially manipulated by DGK overcome zeta-deprivation of T cells (Olenchock, Guo et al, 2006; Moon, Wang et al, 2014) first, evaluating the proliferative capacity of DGK α zeta 139-T during repeated antigen challenge, culturing 139CAR-T cells with U87 vtiii for 96 hours, then replanting U87 vtiii, and measuring the proliferative capacity of cells of DGK-T (fig. 39) by using celraray distribution and cell count to measure the proliferative capacity of fasa-T cells (figure 39) as opposed to non-proliferating CARs 9-T-c, and showing that the increased proliferation of FAS-T cells upon exposure to fasy-T-cell activation, increased proliferation of fasy, aak-T cell proliferation when cell proliferation-T cell proliferation-T-cell proliferation-proliferation of fasy, no-proliferation of fasy, fave cell proliferation-proliferation of fa.
Example 5 reprogramming of DGK-manipulated T cells into Effector memory T cells
Loss of both DGK α and DGK ζ was reported to differentiate CD 8T cells into short-lived effector cells and effector memory groups (Yang, Zhang et al, 2016) to investigate whether DGK deficiency alters T cell differentiation, the characteristics of memory subpopulations of DGK-manipulated CAR-T cells when DGK-manipulated 139CAR-T cells were co-cultured with U87vIII for 4 days were demonstrated DGK-manipulated CAR-T cells that showed less than AAVS 1139 CAR-T cells prior to tumor inoculation (fig. 43) after tumor inoculation for 4 days, DGK α 139CAR-T cells's initial T cell population preferentially differentiated into effector memory cells resulting in smaller initial T cell and central memory T cell populations (fig. 43) following which artificial manipulation of DGK reprogrammed T cells at the transcriptional level was investigated as a greater extent of expression of relevant transcription factors after 48 hours of activation of T cells using CD 3/dynabead, CD28, CD-mediated, CAR-T cell population was identified as exhibiting a greater increase in vitro transcriptional control of the effects of CAR-T cell depletion by the artificial T-effector cells when DGK-effector cells were expressed by the artificial knock-effector cells, CD-T cell line, CD-T-effector cells, which showed no increase in response to the expression profile of the artificial effector cells, when the artificial effector cells of the expression of the effector cells, CD-effector receptor-effector cells, which was demonstrated by the artificial receptor-effector cells, which was shown by the artificial receptor-effector receptor-T receptor-effector cell line, which was increased by the artificial receptor-effector cell line, which was shown by the artificial receptor-effector cell line, which was increased after 48 hours, and was not expressed by the artificial receptor-effector cell line, and was shown by the artificial receptor-effector cell line, and was increased.
Example 6 tumor infiltration and tumor removal Effect of DGK-engineered T cells
To study the in vivo functional relevance of enhanced effector function of DGK α ζ 139CAR-T, AAVS 1139 CAR-T or DGK α ζ 139CAR-T was injected Intravenously (IV) or Intratumorally (IT) into U87vIII transplanted NSG mice the effect of adoptive cell transfer may differ significantly depending on the number of tumors present and the number of immune cells Levi J, Rupp et al demonstrated in experiments with mice with low tumor volumes that high T cell injection was able to completely eliminate tumors in both control groups of CD19 CAR-T cells and PD1 knockout CD19 CAR-T cells (Rupp, Schumann et al, 2017.) thus, this study used low T cell volumes in a high tumor volume model to study the in vivo efficacy of DGK knockout T cells3First injection of T cells; when the tumor volume reaches 400 +/-50 mm3It is taken for 4 daysHere, the first and second injections were performed at E: T ratios of approximately 1:10 and 1:20, respectively. numerous studies reported that treatment without Temozolomide (TMZ) during anti-EGFRvIII glioblastoma targeted T cell therapy generally showed ineffective results (Ohno, Ohkuri et al, 2013; Johnson, Scholler et al, 2015), so temozolomide adjuvant was injected intraperitoneally during the second T cell injection to stimulate tumor regression, showing a delay in tumor growth per IV injection group 32 days after TMZ treatment, but compared to the control T cell mouse group, mice injected with AAVS 1139-T did not show anti-tumor effect (fig. 46), whereas the tumors were found completely in DGK α CAR 139-T mouse group on day 56, although the intratumoral injection of AAVS 1139-T failed to eliminate CAR U vIII, but the results of tumor regression of k α CAR 139-T mouse showed meaningful numbers of tumor regression, 52, for the days of T cell infiltration, 139-T139 and 52, for meaningful tumor cell counts.
In order to additionally characterize the in vivo function of DGK knockout 139CAR-T cells, the viability of injected AAVS 1139 CAR-T cells, α KO 139CAR-T cells, ζ KO 139CAR-T cells, and dKO 139CAR-T cells was analyzed the results identified that ζ KO 139CAR-T cells and dKO 139CAR-T cells maintained significantly large numbers in tumors (fig. 47) because dividing T cells (represented by Ki-67 stained cells) were present in larger numbers, identified as functionally dominant effector T cells with increased T-beta expression and enhanced secretion capacity of cytokines such as IFN- γ and TNF- α (fig. 48).
Taken together, the data results demonstrate that manual manipulation of DGK by CRIPSR/Cas9 can enhance the in vivo anti-tumor efficacy of human CAR-T cells.
Industrial applicability
An effective immune cell therapeutic agent can be obtained by using modified immune cells (containing artificial modified immune regulatory genes and artificial receptors). For example, when immune cells manipulated by immunization with the composition for immune cell manipulation of the present invention are used, they can be used as effective immune cell therapeutic agents since they can improve the immune efficacy in terms of survival, proliferation, persistence, cytotoxicity, cytokine release and/or infiltration, etc., of immune cells that are capable of specifically binding to a specific antigen.
[ CHARACTERS OF THE SEQUENCE ]
Target sequence of immune regulatory gene
Sequence listing
<110> Turkin (ToolGen Incorporation)
<120> immune cells manipulated by human
<130>OPP17-041-NP-PCT-PCT
<150>US 62/502,822
<151>2017-05-08
<150>PCT/KR 2017/008835
<151>2017-08-14
<150>US 62/595159
<151>2017-12-06
<160>289
<170>SIPOSequenceListing 1.0
<210>1
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>1
cttgtggcgc tgaaaacgaa cgg 23
<210>2
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>2
atgccacttc tcagtacatg tgg 23
<210>3
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>3
gccacttctc agtacatgtg ggg 23
<210>4
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>4
gccccacatg tactgagaag tgg 23
<210>5
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>5
tcagtacatg tggggcgttc agg 23
<210>6
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>6
gggcgttcag gacacagact tgg 23
<210>7
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>7
cacagacttg gtactgagga agg 23
<210>8
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>8
ggcgctgttc agcacgctca agg 23
<210>9
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>9
cacgcaactt taaattccgc tgg 23
<210>10
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>10
cggggctttg ctatgatact cgg 23
<210>11
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>11
ggcttccaca gacacaccca tgg 23
<210>12
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>12
tgaagtccac ttcgggccat ggg 23
<210>13
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>13
ctgtacgaca cggacagaaa tgg 23
<210>14
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>14
tgtacgacac ggacagaaat ggg 23
<210>15
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>15
cacggacaga aatgggatcc tgg 23
<210>16
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>16
gatgcgagtg gctgaatacc tgg 23
<210>17
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>17
gagtggctga atacctggat tgg 23
<210>18
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>18
agtggctgaa tacctggatt ggg 23
<210>19
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>19
attgggatgt gtctgagctg agg 23
<210>20
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>20
atgaaagagattgactatga tgg 23
<210>21
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>21
ctctgtctct caagctgagt ggg 23
<210>22
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>22
tctctcaagc tgagtgggtc cgg 23
<210>23
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>23
ctctcaagct gagtgggtcc ggg 23
<210>24
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>24
caagctgagt gggtccgggc tgg 23
<210>25
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>25
ttgacatgac tggagagaag agg 23
<210>26
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>26
gactggagag aagaggtcgt tgg 23
<210>27
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>27
gagacgggag caaagctgct ggg 23
<210>28
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>28
agagacggga gcaaagctgc tgg 23
<210>29
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>29
tggtttctag gtgcagagac ggg 23
<210>30
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>30
taagtgaagg tctggtttct agg 23
<210>31
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>31
tgcccatgta agtgaaggtc tgg 23
<210>32
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>32
gaacttgccc atgtaagtga agg 23
<210>33
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>33
tccattgacc ctcagtaccc tgg 23
<210>34
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>34
tatgccttct gggtagcagc tgg 23
<210>35
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>35
tgagtgcagg catcttgcaa ggg 23
<210>36
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>36
gagtgcaggc atcttgcaag ggg 23
<210>37
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>37
gatgaggctg tggttgaagc tgg 23
<210>38
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>38
ccactggcca caggacccct ggg 23
<210>39
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>39
gggacatggt gcacacaccc agg 23
<210>40
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>40
gagtacaggt ggtccaggtc agg 23
<210>41
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>41
gcggagagta caggtggtcc agg 23
<210>42
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>42
gcggtggcgg agagtacagg tgg 23
<210>43
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>43
tctcctgcac agccagaata agg 23
<210>44
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>44
acgcagaagg gtcctggtag agg 23
<210>45
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>45
aggtggtggg taggccagag agg 23
<210>46
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>46
cccaagccag ccacggaccc agg 23
<210>47
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>47
acctgggtcc gtggctggct tgg 23
<210>48
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>48
aagagacctg ggtccgtggc tgg 23
<210>49
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>49
ggatcattgg gaagagacct ggg 23
<210>50
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>50
gggatcattg ggaagagacc tgg 23
<210>51
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>51
caggatagtc tgggatcatt ggg 23
<210>52
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>52
ggaaagaatc caggatagtc tgg 23
<210>53
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>53
cagtgccaga gagacctaca tgg 23
<210>54
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>54
ctgtaccatg taggtctctc tgg 23
<210>55
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>55
agagacctac atggtacagc tgg 23
<210>56
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>56
ctgggccagc tgtaccatgt agg 23
<210>57
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>57
agggaaaggg cttacggtct ggg 23
<210>58
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>58
cagggaaagg gcttacggtc tgg 23
<210>59
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>59
tctggagatc ttcttgcaac agg 23
<210>60
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>60
ctccggttca tgactttgaa agg 23
<210>61
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>61
gtcttccatc ttcgtctttc agg 23
<210>62
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>62
gaagacttcg agacccattt agg 23
<210>63
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>63
tcgagaccca tttaggatca cgg 23
<210>64
<211>23
<212>DNA
<213> Intelligent (HomoSapiens)
<400>64
gtagcgccgt gatcctaaat ggg 23
<210>65
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>65
cgtagcgccg tgatcctaaa tgg 23
<210>66
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>66
catttaggat cacggcgcta cgg 23
<210>67
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>67
ggtcccaata ttgaagccca tgg 23
<210>68
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>68
gatccatggg cttcaatatt ggg 23
<210>69
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>69
agatccatgg gcttcaatat tgg 23
<210>70
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>70
gcttctacca taagatccat ggg 23
<210>71
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>71
cgcttctacc ataagatcca tgg 23
<210>72
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>72
gcatttgcaa aaattcgccg tgg 23
<210>73
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>73
atgacctgag aattaattta tgg 23
<210>74
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>74
ccatgcactc ccagacatcg tgg 23
<210>75
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>75
gcactggtgc gggtggaact cgg 23
<210>76
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>76
acacgttgca ctggtgcggg tgg 23
<210>77
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>77
cgaacacgtt gcactggtgc ggg 23
<210>78
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>78
acgaacacgt tgcactggtg cgg 23
<210>79
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>79
tgtagacgaa cacgttgcac tgg 23
<210>80
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>80
gcgcatgtca cacaggcgga tgg 23
<210>81
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>81
aggagcgcat gtcacacagg cgg 23
<210>82
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>82
ccgaggagcg catgtcacac agg 23
<210>83
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>83
cctgtgtgac atgcgctcct cgg 23
<210>84
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>84
cgactggcca gggcgcctgt ggg 23
<210>85
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>85
accgcccaga cgactggcca ggg 23
<210>86
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>86
caccgcccag acgactggcc agg 23
<210>87
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>87
gtctgggcgg tgctacaact ggg 23
<210>88
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>88
ctacaactgg gctggcggcc agg 23
<210>89
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>89
cacctaccta agaaccatcc tgg 23
<210>90
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>90
cggtcaccac gagcagggct ggg 23
<210>91
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>91
gccctgctcg tggtgaccga agg 23
<210>92
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>92
cggagagctt cgtgctaaac tgg 23
<210>93
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>93
cagcttgtcc gtctggttgc tgg 23
<210>94
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>94
aggcggccag cttgtccgtc tgg 23
<210>95
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>95
ccgggctggc tgcggtcctc ggg 23
<210>96
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>96
cgttgggcag ttgtgtgaca cgg 23
<210>97
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>97
cataaagcca tggcttgcct tgg 23
<210>98
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>98
ccttggattt cagcggcaca agg 23
<210>99
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>99
ccttgtgccg ctgaaatcca agg 23
<210>100
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>100
cactcacctt tgcagaagac agg 23
<210>101
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>101
ttccatgcta gcaatgcacg tgg 23
<210>102
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>102
ggccacgtgc attgctagca tgg 23
<210>103
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>103
ggcccagcct gctgtggtac tgg 23
<210>104
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>104
aggtccgggt gacagtgctt cgg 23
<210>105
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>105
ccgggtgaca gtgcttcggc agg 23
<210>106
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>106
ctgtgcggca acctacatga tgg 23
<210>107
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>107
caactcattc cccatcatgt agg 23
<210>108
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>108
ctagatgatt ccatctgcac ggg 23
<210>109
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>109
ggctaggagt cagcgacata tgg 23
<210>110
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>110
gctaggagtc agcgacatat ggg 23
<210>111
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>111
ctaggagtca gcgacatatg ggg 23
<210>112
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>112
gtactgtgta gccaggatgc tgg 23
<210>113
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>113
acgagcactc accagcatcc tgg 23
<210>114
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>114
aggctccagg aatgtccgcg agg 23
<210>115
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>115
acttacctcg cggacattcc tgg 23
<210>116
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>116
caccctgggc acttacctcg cgg 23
<210>117
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>117
gtgccgtaca aaggttggct ggg 23
<210>118
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>118
ggtgccgtac aaaggttggc tgg 23
<210>119
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>119
ctctcctcag taccacagca agg 23
<210>120
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>120
cctggggcct ccgggcgcgg agg 23
<210>121
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>121
agtactcacc tggggcctcc ggg 23
<210>122
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>122
agggtctcca gcggccctcc tgg 23
<210>123
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>123
gcaagtactt acgcctcctt ggg 23
<210>124
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>124
ttgcggtaca tctccagcct ggg 23
<210>125
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>125
tttgcggtac atctccagcc tgg 23
<210>126
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>126
gcaaaacctg tccactctta tgg 23
<210>127
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>127
ttggtgccat aagagtggac agg 23
<210>128
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>128
ggtgcaagtt tcttatatgt tgg 23
<210>129
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>129
acctgatgca tataataatc agg 23
<210>130
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>130
acctgattat tatatgcatc agg 23
<210>131
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>131
cagagcacca gagtgccgtc tgg 23
<210>132
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>132
agagcaccag agtgccgtct ggg 23
<210>133
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>133
agagtgccgt ctgggtctga agg 23
<210>134
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>134
aggaaggccg tccattctca ggg 23
<210>135
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>135
ggatagaacc aaccatgttg agg 23
<210>136
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>136
tctgttgccc tcaacatggt tgg 23
<210>137
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>137
ttagtctgtt gccctcaaca tgg 23
<210>138
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>138
gtctggcaaa tgggaggtga tgg 23
<210>139
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>139
cagaggttct gtctggcaaa tgg 23
<210>140
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>140
ttgtagccag aggttctgtc tgg 23
<210>141
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>141
acttctggat gagctctctc agg 23
<210>142
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>142
agagctcatc cagaagtaaa tgg 23
<210>143
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>143
ttggtgtctc catttacttc tgg 23
<210>144
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>144
ttctggcttc ccttcataca ggg 23
<210>145
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>145
caggactcac acgactattc tgg 23
<210>146
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>146
ctactttctt gtgtaaagtc agg 23
<210>147
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>147
gactttacac aagaaagtag agg 23
<210>148
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>148
gtctttctcc attagccttt tgg 23
<210>149
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>149
aatggagaaa gacgtaactt cgg 23
<210>150
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>150
atggagaaag acgtaacttc ggg 23
<210>151
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>151
tggagaaaga cgtaacttcg ggg 23
<210>152
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>152
tttggttgac tgctttcacc tgg 23
<210>153
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>153
tcactcaaat cggagacatt tgg 23
<210>154
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>154
atctgaagct ctggattttc agg 23
<210>155
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>155
gcttcagatt ctgaatgagc agg 23
<210>156
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>156
cagattctga atgagcagga ggg 23
<210>157
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>157
aaggcagtgc taatgcctaa tgg 23
<210>158
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>158
gcagaaactg tagcaccatt agg 23
<210>159
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>159
accgcaatgg aaacacaatc tgg 23
<210>160
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>160
tgtggttttc tgcaccgcaa tgg 23
<210>161
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>161
cataaatgcc attaacagtc agg 23
<210>162
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>162
attagtagcc tgactgttaa tgg 23
<210>163
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>163
cgatgggtga gtgatctcac agg 23
<210>164
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>164
actcacccat cgcatacctc agg 23
<210>165
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>165
ctcacccatc gcatacctca ggg 23
<210>166
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>166
agcaacagga ggagttgcag agg 23
<210>167
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>167
ccagtaggat cagcaacagg agg 23
<210>168
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>168
ctcctgttgc tgatcctact ggg 23
<210>169
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>169
ggcccagtag gatcagcaac agg 23
<210>170
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>170
ttgctgatcc tactgggccc tgg 23
<210>171
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>171
tggcaacagc ttgcagctgt ggg 23
<210>172
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>172
cttgggtccc ctgcttgccc ggg 23
<210>173
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>173
gtcccctgct tgcccgggac cgg 23
<210>174
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>174
ctccggtccc gggcaagcag ggg 23
<210>175
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>175
tctccggtcc cgggcaagca ggg 23
<210>176
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>176
gtctccggtc ccgggcaagc agg 23
<210>177
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>177
gcttgcccgg gaccggagac agg 23
<210>178
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>178
ggtggcctgt ctccggtccc ggg 23
<210>179
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>179
cggtggcctg tctccggtcc cgg 23
<210>180
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>180
catattcggt ggcctgtctc cgg 23
<210>181
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>181
atctaggtac tcatattcgg tgg 23
<210>182
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>182
ataatctagg tactcatatt cgg 23
<210>183
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>183
ttatgatttc ctgccagaaa cgg 23
<210>184
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>184
atttctggag gctccgtttc tgg 23
<210>185
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>185
actgacacca ctcctctgac tgg 23
<210>186
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>186
ctgacaccac tcctctgact ggg 23
<210>187
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>187
accactcctc tgactgggcc tgg 23
<210>188
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>188
aacccctgag tctaccactg tgg 23
<210>189
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>189
ctccacagtg gtagactcag ggg 23
<210>190
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>190
gctccacagt ggtagactca ggg 23
<210>191
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>191
ggctccacag tggtagactc agg 23
<210>192
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>192
cctgctgcaa ggcgttctac tgg 23
<210>193
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>193
ccagtagaac gccttgcagc agg 23
<210>194
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>194
cgttctactg gcctggatgc agg 23
<210>195
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>195
tctactggcc tggatgcagg agg 23
<210>196
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>196
ccacggagct ggccaacatg ggg 23
<210>197
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>197
cgtggacagg ttccccatgt tgg 23
<210>198
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>198
gtccacggat tcagcagcta tgg 23
<210>199
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>199
gaccactcaa ccagtgccca cgg 23
<210>200
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>200
ggagtggtct gtgcctccgt ggg 23
<210>201
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>201
ggcacagaca actcgactga cgg 23
<210>202
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>202
gacaactcga ctgacggcca cgg 23
<210>203
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>203
aactcgactg acggccacgg agg 23
<210>204
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>204
cacagaaccc agtgccacag agg 23
<210>205
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>205
ggtagtaggt tccatggaca ggg 23
<210>206
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>206
tggtagtagg ttccatggac agg 23
<210>207
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>207
tcttttggta gtaggttcca tgg 23
<210>208
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>208
atggaaccta ctaccaaaag agg 23
<210>209
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>209
aacagacctc ttttggtagt agg 23
<210>210
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>210
gggtatgaac agacctcttt tgg 23
<210>211
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>211
tgtgtcctct gttactcaca agg 23
<210>212
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>212
gtgtcctctg ttactcacaa ggg 23
<210>213
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>213
gtagttgacg gacaaattgc tgg 23
<210>214
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>214
tttgtccgtc aactacccag tgg 23
<210>215
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>215
ttgtccgtca actacccagt ggg 23
<210>216
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>216
tgtccgtcaa ctacccagtg ggg 23
<210>217
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>217
gtccgtcaac tacccagtgg ggg 23
<210>218
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>218
ctctgtgaag cagtgcctgc tgg 23
<210>219
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>219
cctgctggcc atcctaatct tgg 23
<210>220
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>220
ccaagattag gatggccagc agg 23
<210>221
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>221
ggccatccta atcttggcgc tgg 23
<210>222
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>222
caccagcgcc aagattagga tgg 23
<210>223
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>223
agtgcacacg aagaagatag tgg 23
<210>224
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>224
tatcttcttc gtgtgcactg tgg 23
<210>225
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>225
cttcgtgtgc actgtggtgc tgg 23
<210>226
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>226
ggcggtccgc ctctcccgca agg 23
<210>227
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>227
gcggtccgcc tctcccgcaa ggg 23
<210>228
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>228
aattacgcac ggggtacatg tgg 23
<210>229
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>229
tgggggagta attacgcacg ggg 23
<210>230
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>230
gtgggggagt aattacgcac ggg 23
<210>231
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>231
ggtgggggag taattacgca cgg 23
<210>232
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>232
taattactcc cccaccgaga tgg 23
<210>233
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>233
agatgcagac catctcggtg ggg 23
<210>234
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>234
gagatgcaga ccatctcggt ggg 23
<210>235
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>235
tgagatgcag accatctcgg tgg 23
<210>236
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>236
ggatgagatg cagaccatct cgg 23
<210>237
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>237
atctcatccc tgttgcctga tgg 23
<210>238
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>238
tcatccctgt tgcctgatgg ggg 23
<210>239
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>239
ctcaccccca tcaggcaaca ggg 23
<210>240
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>240
gagggcccct cacccccatc agg 23
<210>241
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>241
gggccctctg ccacagccaa tgg 23
<210>242
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>242
ccctctgcca cagccaatgg ggg 23
<210>243
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>243
cccccattgg ctgtggcaga ggg 23
<210>244
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>244
gcccccattg gctgtggcag agg 23
<210>245
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>245
ggacaggccc ccattggctg tgg 23
<210>246
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>246
ccgggctctt ggccttggac agg 23
<210>247
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>247
ctgtccaagg ccaagagccc ggg 23
<210>248
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>248
tggcgtcagg cccgggctct tgg 23
<210>249
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>249
cgggcctgac gccagagccc agg 23
<210>250
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>250
caacaaccat gctgggcatc tgg 23
<210>251
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>251
gagggtccag atgcccagca tgg 23
<210>252
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>252
catctggacc ctcctacctc tgg 23
<210>253
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>253
agggctcacc agaggtagga ggg 23
<210>254
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>254
ggagttgatg tcagtcactt ggg 23
<210>255
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>255
tggagttgat gtcagtcact tgg 23
<210>256
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>256
agtgactgac atcaactcca agg 23
<210>257
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>257
gtgactgaca tcaactccaa ggg 23
<210>258
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>258
actccaaggg attggaattg agg 23
<210>259
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>259
cttcctcaat tccaatccct tgg 23
<210>260
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>260
tacagttgag actcagaact tgg 23
<210>261
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>261
ttggaaggcc tgcatcatga tgg 23
<210>262
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>262
agaattggcc atcatgatgc agg 23
<210>263
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>263
gacagggctt atggcagaat tgg 23
<210>264
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>264
tgtaacatac ctggaggaca ggg 23
<210>265
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>265
gtgtaacata cctggaggac agg 23
<210>266
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>266
cgtacctgtg caactcctgt tgg 23
<210>267
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>267
gatctactgg aattcctaat ggg 23
<210>268
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>268
gagtcagctg ttggcccatt agg 23
<210>269
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>269
ctgcctacaa actcagtctc tgg 23
<210>270
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>270
gggcaggcag gacggactcc agg 23
<210>271
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>271
ggagtccgtc ctgcctgccc tgg 23
<210>272
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>272
gagtccgtcc tgcctgccct ggg 23
<210>273
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>273
gaaaagggtc cattggccaa agg 23
<210>274
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>274
gcctgcagaa aagggtccat tgg 23
<210>275
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>275
ttgatgtgct acagggaaca tgg 23
<210>276
<211>23
<212>DNA
<213> Intelligent (HomoSapiens)
<400>276
agcgttcttg atgtgctaca ggg 23
<210>277
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>277
cagcgttctt gatgtgctac agg 23
<210>278
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>278
ctgtagcaca tcaagaacgc tgg 23
<210>279
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>279
tgtagcacat caagaacgct ggg 23
<210>280
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>280
ataggcaata atcatataac agg 23
<210>281
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>281
agtgcgtttc gctgcaggta agg 23
<210>282
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>282
gagtgagtgc gtttcgctgc agg 23
<210>283
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>283
gtcaggtttg tgcggttatg agg 23
<210>284
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>284
cgctgctggt caggtttgtg cgg 23
<210>285
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>285
aaacctgacc agcagcgcag agg 23
<210>286
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>286
ccagcagcgc agaggagccg tgg 23
<210>287
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>287
ccacggctcc tctgcgctgc tgg 23
<210>288
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>288
ccaactatct aactccactc agg 23
<210>289
<211>23
<212>DNA
<213> Intelligent (Homo Sapiens)
<400>289
cctgagtgga gttagatagt tgg 23

Claims (70)

1. A composition for manipulating immune cells, the composition comprising:
a guide nucleic acid capable of forming a complementary binding to a target sequence in the nucleic acid sequence of at least one immunomodulatory gene selected from the group consisting of: PD-1 gene, CTLA-4 gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene, PPP2r2d gene, TET2 gene, PSGL-1 gene, A20 gene and KDM6A gene; and
an artificial receptor, which is an artificially prepared receptor and is not a wild-type receptor.
2. The composition for manipulation of an immune cell according to claim 1, further comprising at least one editing protein selected from the group consisting of: a Cas9 protein derived from streptococcus pyogenes, a Cas9 protein derived from campylobacter jejuni, a Cas9 protein derived from streptococcus thermophilus, a Cas9 protein derived from staphylococcus aureus, a Cas9 protein derived from neisseria meningitidis, and a Cpf1 protein.
3. The composition for manipulation of an immune cell according to claim 1, wherein the target sequence is a contiguous 10bp-25bp nucleotide sequence located in the promoter region of the immune modulatory gene.
4. The composition for manipulation of an immune cell according to claim 1, wherein the target sequence is a contiguous 10bp-25bp nucleotide sequence located in an intron region of the immune modulatory gene.
5. The composition for manipulation of an immune cell according to claim 1, wherein the target sequence is a contiguous 10bp-25bp nucleotide sequence located in an exon region of the immune modulatory gene.
6. The composition for manipulation of an immune cell according to claim 1, wherein the target sequence is a contiguous 10bp-25bp nucleotide sequence located in an enhancer region of the immune modulatory gene.
7. The composition for manipulation of an immune cell according to claim 1, wherein the target sequence is a contiguous 10bp-25bp nucleotide sequence located in the 3'-UTR (untranslated region) or the 5' -UTR of the immunomodulatory gene.
8. The composition for manipulation of an immune cell according to claim 1, wherein the target sequence is a contiguous nucleotide sequence of 10bp to 25bp at the 5 'end and/or 3' end of a PAM (pro-spacer sequence adjacent motif) sequence in the nucleic acid sequence adjacent to the immune regulatory gene.
9. The composition for manipulation of immune cells according to claim 8, wherein the PAM sequence is at least one sequence selected from the group consisting of:
5'-NGG-3' (N is A, T, C or G);
5 '-NNRYAC-3' (N is each independently A, T, C or G; R is A or G; Y is C or T);
5'-NNAGAAW-3' (N is each independently A, T, C or G; W is A or T);
5'-NNNNGATT-3' (N is each independently A, T, C or G);
5'-NNGRR (T) -3' (N is each independently A, T, C or G; R is A or G; Y is C or T); and
5'-TTN-3' (N is A, T, C or G).
10. The composition for manipulation of an immune cell according to claim 1, wherein the target sequence is a sequence selected from the group consisting of SEQ ID NOs: 1-SEQ ID NO: 289.
11. The composition for manipulation of an immune cell according to claim 1, wherein the guide nucleic acid comprises a guide domain capable of forming a complementary binding to a target sequence on the immunomodulatory gene, wherein the complementary binding may comprise 0-5 mismatches.
12. The composition for manipulation of an immune cell according to claim 11, wherein the targeting domain comprises a nucleotide sequence that is complementary to a target sequence on the immunomodulatory gene, wherein complementary nucleotide sequence can comprise 0-5 mismatches.
13. The composition for manipulation of an immune cell according to claim 1, wherein the guide nucleic acid comprises at least one domain selected from the group consisting of: a first complementary domain, a linker domain, a second complementary domain, a proximal domain, and a tail domain.
14. The composition for manipulation of an immune cell according to claim 1, wherein the artificial receptor has binding specificity for at least one antigen.
15. The composition for manipulation of an immune cell according to claim 14, wherein the at least one antigen is an antigen specifically expressed by a cancer cell or/and a virus.
16. The composition for manipulation of an immune cell according to claim 14, wherein said at least one antigen is a tumor associated antigen.
17. The composition for manipulation of an immune cell according to claim 14, wherein the at least one antigen is one or more selected from the group consisting of:
a, ALK, alpha-fetoprotein (AFP), adrenoreceptor 3 (ADRB), -folate receptor, AD034, AKT, BCMA, -human chorionic gonadotropin, B7H (CD276), BST, BRAP, CD79, CD123, CD138, CD160, CD171, CD179, Carbonic Anhydrase IX (CAIX), CA-125, carcinoembryonic antigen (CEA), CCR, C-type lectin-like molecule (CLL-1 or CLECL), claudin (Claudin), CXORF, CAGE, NYX, CLCP-7, CT/HOMP-85, cTAGE-1, ERBB, Epidermal Growth Factor Receptor (EGFR), EGFR type III (EGFRvIII), cell adhesion Molecule (MAG), NYE-like factor 2 (NYF-2), TNF-TEM-85, TNF-CETF-1, MAGE-2-TNF-receptor (MAG-receptor), VEGF-7, VEGF-2-receptor (MAG-7), VEGF-2-TNF-7, VEGF-2-TNF-7, VEGF-2-7, VEGF-related, VEGF-7, VEGF-related, VEGF-7, VEGF-related, VEGF-7, VEGF-related, VEGF-7, VEGF-related, VEGF-7, VEGF-related, VEGF-7.
18. The composition for manipulation of an immune cell according to claim 1, wherein said artificial receptor is a Chimeric Antigen Receptor (CAR).
19. The composition for manipulation of an immune cell according to claim 1, wherein the artificial receptor is a manually manipulated or modified T Cell Receptor (TCR).
20. The composition for manipulation of an immune cell according to claim 1 or 2, wherein the guide nucleic acid, the artificial receptor and the editing protein are in the form of nucleic acid sequences encoding each of them.
21. The composition for manipulation of an immune cell according to claim 20, wherein the nucleic acid sequence is contained in a plasmid or viral vector.
22. The composition for manipulation of an immune cell according to claim 21, wherein the viral vector is one or more selected from the group consisting of: retrovirus, lentivirus, adenovirus, adeno-associated virus (AAV), vaccinia virus, poxvirus, or herpes simplex virus.
23. The composition for manipulation of an immune cell according to claim 1 or 2, wherein the artificial receptor and editing protein are in the form of mrnas encoding each.
24. The composition for manipulation of an immune cell according to claim 1 or 2, wherein the artificial receptor and editing protein are in the form of a polypeptide or protein.
25. The composition for manipulation of an immune cell according to claim 2, wherein the composition is in the form of a guide nucleic acid-editing protein complex.
26. A manipulated immune cell, the manipulated immune cell comprising:
at least one artificially engineered immunomodulatory gene and/or a product expressed by said artificially engineered immunomodulatory gene, said immunomodulatory gene selected from the group consisting of: PD-1 gene, CTLA-4 gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene, PPP2r2d gene, TET2 gene, PSGL-1 gene, A20 gene and KDM6A gene; and;
at least one artificial receptor protein and/or a nucleic acid encoding said artificial receptor protein.
27. The manipulated immune cell of claim 26, wherein the at least one artificially engineered immunomodulatory gene comprises an artificial modification within a nucleotide sequence of the immunomodulatory gene.
28. The manipulated immune cell of claim 26, wherein the at least one artificially engineered immune modulatory gene comprises a deletion and/or insertion of at least one nucleotide in a region of 1bp-50bp nucleotide sequence within or adjacent to the 5 'end and/or 3' end of the target sequence in the immune modulatory gene.
29. The manipulated immune cell of claim 26, wherein the at least one artificially engineered immune modulatory gene comprises a deletion and/or insertion of at least one nucleotide in a contiguous region of nucleotide sequence from 1bp to 50bp proximal to the 5 'end and/or 3' end of a PAM sequence in the nucleic acid sequence of the immune modulatory gene.
30. The manipulated immune cell of claim 28 or 29, wherein the deletion of at least one nucleotide is a continuous 1bp-50bp deletion, a discontinuous 1bp-50bp deletion, or a 1bp-50bp deletion in which a continuous form and a discontinuous form are mixed.
31. The manipulated immune cell of claim 28 or 29, wherein the deletion of the at least one nucleotide is a contiguous deletion of 2bp-50 bp.
32. The manipulated immune cell of claim 28 or 29, wherein the insertion of the at least one nucleotide is a contiguous 1bp-50bp insertion, a non-contiguous 1bp-50bp insertion, or a 1bp-50bp insertion in which a contiguous form and a non-contiguous form are mixed.
33. The manipulated immune cell of claim 28 or 29, wherein the insertion of the at least one nucleotide is an insertion of a contiguous 5bp-1000bp nucleotide fragment.
34. The manipulated immune cell of claim 28 or 29, wherein the insertion of the at least one nucleotide is an insertion of a partial or complete nucleotide sequence of a particular gene.
35. The manipulated immune cell of claim 34, wherein the specific gene is an exogenous gene introduced from an external region, and the immune cell containing the immunomodulatory gene does not contain the exogenous gene.
36. The manipulated immune cell of claim 34, wherein the specific gene is an endogenous gene present in the genome of the immune cell comprising the immunomodulatory gene.
37. The manipulated immune cell of claim 28 or 29, wherein the deletion and insertion of the at least one nucleotide occurs in the same nucleotide sequence region.
38. The manipulated immune cell of claim 28 or 29, wherein the deletion and insertion of the at least one nucleotide occurs in different nucleotide sequence regions.
39. The manipulated immune cell of claim 26, wherein at least one product expressed by the engineered immunomodulatory gene is in the form of mRNA and/or protein.
40. The manipulated immune cell of claim 26, wherein the product expressed by the engineered immunomodulatory gene has a reduced or suppressed expression level as compared to the amount of the product expressed by the immunomodulatory gene of a wild-type immune cell that is not manually manipulated.
41. The manipulated immune cell of claim 40, wherein the non-human wild-type immune cell is an immune cell isolated from a human.
42. The manipulated immune cell of claim 40, wherein the non-manipulated wild-type immune cell is a pre-manipulated immune cell.
43. The manipulated immune cell of claim 26, wherein the nucleic acid encoding the artificial receptor protein is not inserted into the genome of the manipulated immune cell.
44. The manipulated immune cell of claim 26, wherein the nucleic acid encoding the artificial receptor protein is inserted into a 3'-UTR region, a 5' -UTR region, an intron region, an exon region, a promoter region, and/or an enhancer region of the immunomodulatory gene in the genome of the manipulated immune cell.
45. The manipulated immune cell of claim 26, wherein the nucleic acid encoding the artificial receptor protein is inserted into at least one intron selected from introns present in the genome of the manipulated immune cell.
46. The manipulated immune cell of claim 26, wherein the nucleic acid encoding the artificial receptor protein is inserted into at least one exon selected from exons present in the genome of the manipulated immune cell.
47. The manipulated immune cell of claim 26, wherein the nucleic acid encoding the artificial receptor protein is inserted into at least one promoter selected from promoters present in the genome of the manipulated immune cell.
48. The manipulated immune cell of claim 26, wherein the nucleic acid encoding the artificial receptor protein is inserted into at least one enhancer selected from enhancers present in the genome of the manipulated immune cell.
49. The manipulated immune cell of claim 26, wherein the nucleic acid encoding the artificial receptor protein is inserted into one or more regions other than an intron, an exon, a promoter, and an enhancer present in the genome of the manipulated immune cell.
50. The manipulated immune cell of claim 26, wherein the manipulated immune cell is an artificially manipulated immune cell selected from the group consisting of: dendritic cells, T cells, NK cells, NKT cells and CIK cells.
51. The manipulated immune cell of claim 26, which exhibits at least one characteristic:
increased production and/or secretion of cytokines;
cell proliferation, and
increased cytotoxicity.
52. The manipulated immune cell of claim 51, wherein the cytokine is one or more selected from the group consisting of IL-2, TNF α, and IFN- γ.
53. A method for producing a manipulated immune cell, the method comprising contacting:
(a) an immune cell;
(b) an artificial receptor protein or a composition for expressing an artificial receptor protein; and
(c) a composition for gene manipulation capable of manual manipulation of at least one immunomodulatory gene selected from the group consisting of: PD-1 gene, CTLA-4 gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene, PPP2r2d, TET2 gene, PSGL-1 gene, A20 gene and KDM6A gene.
54. The method for producing a manipulated immune cell according to claim 53, wherein (a) the immune cell is an immune cell isolated from a human body or an immune cell differentiated from a stem cell.
55. The method for producing a manipulated immune cell according to claim 53 wherein (b) the composition for expressing an artificial receptor protein comprises a nucleic acid sequence encoding the artificial receptor protein.
56. The method for producing a manipulated immune cell of claim 53, wherein (c) the composition for genetic manipulation comprises:
a guide nucleic acid or a nucleic acid encoding the guide nucleic acid, which is complementary to a target sequence of SEQ ID NO: 1-SEQ ID NO: 289 have homology or are capable of forming complementary binding therewith: PD-1 gene, CTLA-4 gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene, PPP2r2d gene, TET2 gene, PSGL-1 gene, A20 gene and KDM6A gene; and
at least one editing protein, or a nucleic acid encoding the editing protein, selected from the group consisting of: a Cas9 protein derived from streptococcus pyogenes, a Cas9 protein derived from campylobacter jejuni, a Cas9 protein derived from streptococcus thermophilus, a Cas9 protein derived from staphylococcus aureus, a Cas9 protein derived from neisseria meningitidis, and a Cpf1 protein.
57. The method for producing a manipulated immune cell according to claim 56 wherein the guide nucleic acid and the editing protein are each in the form of a nucleotide sequence in at least one vector or in the form of a guide nucleic acid-editing protein complex in which the guide nucleic acid and the editing protein bind.
58. The method for producing a manipulated immune cell according to claim 53 wherein the contacting is performed ex vivo.
59. The method for producing a manipulated immune cell of claim 53,
wherein the contacting is sequentially or simultaneously contacting (a) the immune cell with (b) a composition for expressing an artificial receptor protein and (c) a composition for gene manipulation.
60. The method for producing a manipulated immune cell according to claim 53 wherein the contacting is by at least one method selected from the group consisting of: electroporation, liposomes, plasmids, viral vectors, nanoparticles, and Protein Translocation Domain (PTD) fusion protein methods.
61. A method for treating an immune disease, the method comprising administering to a subject a composition comprising the manipulated immune cell of claim 26 as an active ingredient.
62. A method for treating an immune disease according to claim 61, wherein the composition further comprises one or more selected from the group consisting of: antigen binding agents, cytokines, secretagogues for cytokines, inhibitors of cytokines, and immune checkpoint inhibitors.
63. A method for treating an immune disease according to claim 61, wherein the composition further comprises a suitable carrier for delivering the manipulated immune cells into the body.
64. A method for treatment of an immune disease according to claim 62, wherein the immune checkpoint inhibitor is an inhibitor of PD-1, PD-L1, LAG-3, TIM-3, CTLA-4, TIGIT, BTLA, IDO, VISTA, ICOS, KIR, CD160, CD244 or CD 39.
65. The method for treating an immune disease according to claim 61, wherein the manipulated immune cells are autologous cells of the subject, or allogeneic cells of the subject.
66. A method for treating an immune disorder according to claim 61, wherein the immune disorder is an autoimmune disorder.
67. The method for treating an immune disorder according to claim 66, wherein the autoimmune disorder is graft-versus-host disease (GVHD), systemic lupus erythematosus, celiac disease, type 1 diabetes, graves' disease, inflammatory bowel disease, psoriasis, rheumatoid arthritis, or multiple sclerosis.
68. A method for treating an immune disease according to claim 61, wherein said immune disease is a viral infectious disease, a disease caused by a prion pathogen, or cancer.
69. A method for treating an immune disease according to claim 61, wherein administration of the composition to a subject having an immune disease is by a method selected from injection, infusion, implantation or transplantation.
70. The method of claim 61, wherein the subject is a mammal, including humans, monkeys, mice, and rats.
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